Burkholderia glumae and B. gladioli Cause Bacterial Panicle Blight in Rice in the Southern United States.

Bacterial panicle blight (BPB) is among the three most limiting rice diseases in Louisiana and the southern United States. The identity and characterization of pathogens associated with this disease was unclear. This research details studies carried out on the pathogens causing BPB on rice in Louisiana and other rice producing southern states. Bacterial strains were isolated from BPB-infected sheath, panicle, or grain samples collected from rice fields in Louisiana, Arkansas, Texas, and Mississippi. In greenhouse inoculation tests, 292 of 364 strains were pathogenic on rice seedlings or panicles. Identification of strains in the pathogen complex by growth on S-PG medium, carbon source utilization profile (Biolog), cellular fatty acid analysis, and polymerase chain reaction (PCR) methods revealed that 76 and 5% of the strains were Burkholderia glumae and B. gladioli, respectively. The other strains have not been conclusively identified. Although strains of both species produced similar symptoms on rice, B. glumae strains were generally more aggressive and caused more severe symptoms on rice than B. gladioli. Virulent strains of both species produced toxoflavin in culture. The two species had similar growth responses to temperature, and optima ranged from 38 to 40°C for B. glumae and 35 to 37°C for B. gladioli. PCR was the most sensitive and accurate method tested for identifying the bacterial pathogens to the species level. The 16S rDNA gene and 16S-23S rDNA internal transcribed spacer (ITS) region sequences of the B. glumae and B. gladioli strains from rice showed more than 99% sequence homology with published sequences. A real-time PCR system was developed to detect and quantify this pathogen from infected seed lots. Our results clearly indicate that B. glumae and B. gladioli were the major pathogens causing BPB in the southern United States.

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 Bacterial Soft Rot on Vanda Orchids Caused by Dickeya chrysanthemi (Erwinia chrysanthemi) in the United States.

Vanda orchids are epiphytes grown for their attractive flowers by commercial producers and hobbyists throughout Florida. In August 2007, five Vanda hybrids, with an economic value of $150 each, were found at a nursery in central Florida with leaves that were macerated, brown, and water soaked. According to the growers, the plants were normal the previous day but symptoms developed rapidly. The plants were immediately removed from the greenhouse to prevent potential disease spread. Bacteria were isolated according to the method of Schaad et al. (1). Isolated bacteria grew at 37°C, were gram negative, degraded pectate, and produced phosphatase. MIDI (Sherlock version TSBA 4.10; Microbial Identification 16 System, Newark, DE) (SIM 0.906) identified the bacteria as Erwinia chrysanthemi (Dickeya chrysanthemi Burkholder et al. 1953) Samson et al. 2005. PCR was performed on the 16S rRNA gene (GenBank Accession No. EU526397) with primers 27f (5'-GAGAGTTTGATCCTG GCTCAG-3') and 1495r (5'-TACGGCTACCTTGTTACGA-3') (2). Subsequent DNA sequencing and GenBank search showed the isolated strain is 99% identical to that of Dickeya chrysanthemi. Four leaves each of six Vanda hybrids were inoculated by injecting approximately 150 µl of a bacteria suspension at 1 × 108 CFU/ml into each leaf. One plant was inoculated with water in each of four leaves. Plants were enclosed in plastic bags and returned to the greenhouse under 50% shade at 29°C day and 17°C night temperatures. Within 24 h, soft rot symptoms appeared on inoculated leaves. The water control appeared normal. D. chrysanthemi was reisolated and identified with the above method, thus Koch's postulates were fulfilled. To our knowledge, this is the first report of a soft rot caused by D. chrysanthemi on Vanda hybrids. Because of the popularity and high value of Vanda orchids, proper identification of this rapidly progressing bacterial disease is of great importance for the commercial producer and homeowner alike. References: (1) 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. (2) W. G. Weisburg. J. Bacteriol. 173:697, 1991.

Polyphasic characterization of xanthomonads isolated from onion, garlic and Welsh onion (Allium spp.) and their relatedness to different Xanthomonas species.

Bacterial blight is an emerging disease that affects primarily onion, but also garlic and Welsh onion. The present study was undertaken to characterize the causative xanthomonad(s) by a polyphasic approach using a worldwide collection of 33 bacterial strains. Analysis of 16S rRNA gene sequence similarities indicated that the causal agent belongs to the campestris core in the genus Xanthomonas, which is in agreement with results of phenotypic characterization (analyses of carbon source utilization and fatty acid methyl esters). However, DNA-DNA hybridization, thermal stability of DNA reassociation and fluorescent amplified fragment length polymorphism analysis allowed the causal agent to be identified as a pathovar of Xanthomonas axonopodis.

Bacterial Canker Caused by Clavibacter michiganensis subsp. michiganensis on Greenhouse-Grown Tomato in the Western Mediterranean Region of Turkey.

During the 2003 winter growing season, a disease occurred on tomato (Lycopersicon esculentum L.) cvs. Selin, Astona, 198 RN, Malike, and Tulin that were growing in almost all greenhouses of Serik, Aksu, Kumluca, Demre, and Kinik in the Antalya Province, and Keçiborlu, Çandir, and Seyhler in the Isparta Province in the western Mediterranean Region of Turkey. Disease incidence ranged from 26 to 65%, resulting in economically heavy losses. Symptoms were water-soaked, dark brown-to-black lesions on the leaf margins and asymmetrical wilting of the leaflets. In advanced stages of disease, vascular tissue had a light brown discoloration. A gram-positive bacterium was consistently isolated and formed light gray colonies with internal light gray flecks approximately 2 to 3 mm in diameter on the modified semiselective medium (SCM) (2). Thirty isolates were identified as Clavibacter michiganensis subsp. michiganensis using polymerase chain reaction (PCR) with primers CMM 5 and CMM 6 on the basis of consistent amplification of a 614-bp DNA fragment (1). The isolates were also identified as C. michiganensis subsp. michiganensis (similarity 0.810 to 0.888%) using fatty acid methyl ester (FAME) analysis and Sherlock Microbial Identification System software (Microbial ID, Newark, DE). A pathogenicity test was done by spraying a 6-week-old tomato seedling (cv. Selin) with the bacterial concentration of 108 CFU/ml in sterile water. Control tomato seedlings were only sprayed with sterile water. The inoculated plants were covered with polyethylene bags for 48 h and kept in a growth chamber at 25°C. Symptoms developed on the inoculated plants within approximately 10 days and were similar to those observed in the greenhouse. No symptoms developed on control plants. The bacterium was reisolated from inoculated plants, and its identity was confirmed by colony type on SCM, PCR, and FAME analysis. Although it has been known to be present previously in Aegean, eastern Mediterranean, and the eastern Anatolia regions of Turkey, to our knowledge, this is the first report of bacterial canker on greenhouse tomatoes in the western Mediterranean Region of Turkey. Occurrence of the disease in almost all greenhouses may be the result of the high relative humidity present and the lack of protective bactericide applications in many greenhouses of the region. References: (1) J. Dreier et al. Phytopathology 85:462, 1995. (2) M. Fatmi and N. W. Schaad. Phytopathology 78:121, 1988.

An Outbreak of Bacterial Speck Caused by Pseudomonas syringae pv. tomato on Tomato Transplants Grown in Commercial Seedling Companies Located in the Western Mediterranean Region of Turkey.

A serious outbreak of a leaf spot disease was observed on tomato transplants grown in commercial seedling companies in southwestern Turkey (Antalya) during the springs of 2002 and 2003. Disease incidence was more severe in the western Mediterranean Region of Turkey. Occurrence of the outbreak resulted in approximately 20 and 25% seedling losses in the springs of 2002 and 2003, respectively. The initial symptoms consisted of pronounced water-soaked, dark brown-to-black spots on young expanding leaves that were 1 to 2 mm in diameter. Later, a number of leaf spots on older leaves enlarged and coalesced, causing leaf desiccation and finally, seedling death. In addition, in 2003 the disease incidence was approximately 5% in 142 commercial greenhouses. Tomato production was unaffected since significant outbreaks did not occur on greenhouse plants. No fruit symptoms were observed. Twenty-six strains were isolated from diseased tomato seedlings and plants from different greenhouses located in different places and all were gram negative and fluorescent on King's B medium. All strains were levan and gelatin liquefaction positive and oxidase and arginine dihydrolase negative. None of the 26 strains utilized erythritol and l-lactate as the sole carbon source (1,2). Fatty acid analysis identified the strains as Pseudomonas syringae pv. tomato with similarity indices ranging from 0.876 to 0.932%. Pathogenicity of the isolates was confirmed on 4-week-old tomato seedlings (cv. Biotek Selin) sprayed with the bacterial suspensions containing 108 CFU/ml of sterile water. Later, a number of leaf spots on the leaves enlarged and coalesced, causing leaf desiccation. Inoculated and control tomato seedlings were covered with polyethylene bags and placed in a growth chamber at 25°C for 48 h and then the bags were removed. Small (1 to 2 mm), water-soaked, dark brown-to-black spots similar to those observed in the greenhouses of commercial seedling companies and commercial greenhouses that produce tomato developed on the young expanding leaves of inoculated plants within 7 to 10 days. No symptoms developed on control plants. The bacterium was reisolated from inoculated plants and identified as a strain of Pseudomonas syringae pv. tomato. Koch's postulates were fulfilled. To our knowledge, this is the first report for the occurrence and outbreak of the bacterial speck disease on tomato transplants in greenhouses of commercial tomato seedling production companies in Turkey. References: (1) D. C. Hildebrand et al. Pages 60-80 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. D. Schaad, ed. The American Phytopathological Society, 1988. (2) J. B. Jones et al. Plant Dis. 70:151, 1986.

Bacterial Spot of Tomato and Pepper Caused by Xanthomonas axonopodis pv. vesicatoria in the Western Mediterranean Region of Turkey.

Xanthomonas axonopodis pv. vesicatoria, causal agent of bacterial spot of tomato (Lycopersicon esculentum L.) and sweet pepper (Capsicum annuum L.) was isolated from tomato and pepper plants in greenhouse production in the Province of Antalya, in southwestern Turkey. Disease incidence was less than 4% of plants observed in 2001 and ranged from 1 to 20% in 2002. Eleven seedling-producing companies and 26 greenhouses that produce tomato and pepper were surveyed during the rainy seasons of 2001 and 2002. The increase in disease incidence in 2002 is an indication that this disease is becoming more prevalent on tomato and pepper plants grown in greenhouses in southwestern Turkey. A gram-negative bacterium producing yellow-pigmented colonies on nutrient agar was consistently isolated from brown, circular spots on leaflets of tomato and sweet pepper seedlings. Five isolates were pathogenic on commercial cultivars of tomato and pepper when bacterial suspensions (108 CFU/ml) were infiltrated into the intercellular spaces of leaves to determine race by using procedures described by Bouzar et al. (1). All the isolates produced hypersensitive reaction responses on tomato genotype cv. Hawaii 7998 and pepper genotype cvs. 20 R and 30 R and were designated tomato race 1 pepper race 1 (T1P1) (1). Fatty acid analysis of the strains identified them as X. axonopodis vesicatoria with similarity index values of 0.872 to 0.933. In addition, the strains were tested with X. axonopodis vesicatoria-specific polymerase chain reaction primers (RST 2/3 and RST 9/10) (2). The isolates were determined to be X. axonopodis vesicatoria. Although bacterial spot of tomato has been suspected in Turkey for a number of years, to our knowledge, this is the first report of the bacterium on tomato. References: (1) H. Bouzar et al. Phytopathology 84:663, 1994. (2) R. P. Leite, Jr. et al. Appl. Env. Microbiol. 60:1068, 1994.

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 Leaf Blight of Onion Caused by Xanthomonas campestris in the Continental United States.

In March 1998, a leaf blight of onion (Allium cepa L. '1015') was found on many plants in a plot on the Texas A&M Agricultural Experiment Station in Weslaco. The symptoms were longitudinal chlorotic areas on one side of the leaf, containing sunken, elliptical necrotic lesions. Affected leaves ultimately died. Chlorotic lesions were swabbed with 70% ethanol, and tissue from beneath the epidermis was placed in a drop sterile water for 20 min. Drops were streaked on nutrient agar and incubated at 30°C. Isolations yielded gram-negative, rod-shaped bacteria that formed dark yellow, gummy colonies on yeast dextrose carbonate agar medium, hydrolyzed starch, and had a single, polar flagellum. Analysis of fatty acid methyl ester (FAME) profiles, using the Microbial Identification System (MIS, version 4.15; Microbial Identification, Newark, DE), done at the Texas Plant Disease Diagnostic Laboratory, College Station, identified nine isolates as Xanthomonas campestris (similarity indices of 0.31 to 0.54). Tests at the University of Florida supported this identification: FAME profiles using MIS version 3.9 gave similarity indices of 0.89 to 0.95, and profiles using Biolog GN Microplates, MicroLog database release 3.50 (Biolog, Hayward, CA), gave similarity indices of 0.03 to 0.76. Leaves (15 to 20 cm long) of potted onions (cv. 1015 at the five- to six-leaf stage) were infiltrated with a suspension of bacteria (107 CFU per ml), using a needle and syringe. Plants were maintained in mist chamber in a greenhouse at 24°C. Water-soaking and development of pale green color of the inoculated leaf occurred after 2 days, followed by death after 4 days. There were no symptoms on leaves inoculated with sterile water. Pathogenicity tests on four isolates were repeated once. Bacteria were reisolated on nutrient agar from symptomatic tissue but not from controls. In the field plot, disease severity did not increase as season progressed nor were there any symptoms on bulbs. Symptoms were not observed on onion during the 1999 season. X. campestris was first reported on onion from Hawaii (1). This is the first report of this pathogen on onion in the continental United States. Reference: (1) A. M. Alvarez et al. Phytopathology 68:1132, 1978.