Plant-Pathogenic Acidovorax Species

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CHAPTER

8

Acidovorax cattleyae: Bacterial Brown Spot of Orchids JA AP D. JANSE Department of Laboratory Methods and Diagnostics, Dutch General Inspection Service, Emmeloord, The Netherlands

BACKGROUND

several species, including P. avenae and P. cattleyae, formerly classified in the genus Pseudomonas, in the new genus Acidovorax. P. cattleyae was placed in the newly described species Acidovorax avenae as subsp. cattleyae, together with subsp. avenae (pathogenic on oat) and subsp. citrulli (pathogenic on watermelon and other cucurbits). In 2008, based on DNA-DNA hybridization, 16S rRNA and 16S-23S rRNA internal transcribed spacer (ITS) sequence analysis, amplified fragment length polymorphism (AFLP), and phenotypic traits, Schaad et al. (2008) raised the three subspecies of A. avenae to the species level, and A. avenae subsp. cattleyae was renamed A. cattleyae. The currently accepted taxonomic position, name, and nomenclature for the pathogen that causes brown spot of orchids are Acidovorax cattleyae (cat.tle’ya.e. N.L. gen. n. cattleyae, of Cattleya, a genus of orchid plants). Basonym: Pseudomonas cattleyae (Pavarino, 1911) Savulescu, 1947 (Approved Lists 1980). Other homotypic synonym: Acidovorax avenae subsp. cattleyae (Pavarino, 1911) Willems et al., 1992. Class Betaproteobacteria, family Comamonadaceae. The type strain is Acidovorax cattleyae (Pavarino) Schaad et al. ATCC 33619, which has been deposited in other collections as NCPPB 961, CCUG 21975, ICMP 2826, LMG 2364, LMG 5286, NRRL B-835, and PC 21. The sequence accession number in EMBL and GenBank (16S rRNA gene) for the type strain is AF137504 (GU339087). This strain should be used as a reference in diagnostic tests. In a recent taxonomic study of known Acido­ vorax spp. (Vaneechoutte et al., 2013), the most

■ A bacterial leaf spot and bud rot of orchids was first described by Pavarino (1911) in Italy. Pavarino named one of the four bacterial species isolated from diseased plants Bacterium cattleyae. Although this orchid disease has been observed and reported regularly over the years from orchid production systems (open fields with or without shielding, and greenhouses), private residences and greenhouses, by orchid producers and orchid hobby organizations, very little research has been published on this disease. The causal organism was comprehensively described for the first time in 1946 by Ark and Thomas in California, U.S.A., as Phytomonas cattleyae, causing bacterial leaf spot and bud rot of orchids (Ark and Thomas, 1946). The disease sometimes caused heavy losses, and the causal agent was similar to the bacterium described as Bacterium cattleyae by Pavarino in 1911. In a taxonomic study on the genus Phytomonas, Savulescu (1947) changed Phytomonas cattleyae to Pseudomonas cattleyae. While the exact origin of the pathogen and the disease is unknown, it is possible that the bacterium may have originated in Southeast Asian or South/Central American countries, where the orchid species originated and where the pathogen might have been distributed with breeding and/ or planting material (seedlings, mature plants) around the world. On the basis of DNA-DNA hybridization, polyacrylamide gel electrophoresis (PAGE), and carbon substrate utilization studies, Willems et al. (1992) placed

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CHAPTER 8

closely related plant-pathogenic Acidovorax species to A. cattleyae were A. avenae (from oat), A. citrulli (from watermelon), and A. oryzae (from rice). The other plant-pathogenic Acidovorax species, A. anthurii (from Anthurium), A. konjaci (from Amorphophallus rivieri), and A. valerianellae (from Valerianella locusta and tea), were more distantly related. For more details regarding taxonomic relationships with other plant-pathogenic, environmental, and clinical Acidovorax spp., see Chapter 2 in this book and Vaneechoutte et al. (2013).

HOST RANGE AND GEOGRAPHICAL DISTRIBUTION OF A. CATTLEYAE ■■ The natural host range of A. cattleyae is limited to orchid species and their hybrids. The following hosts were reported in the literature: Cattleya spp., Phalaenopsis spp., and Paphiopedilum (Venus’ slipper) spp., and hybrids of these Phalaenopsis spp. appear to be the most susceptible and sensitive to infection by A. cattleyae (for references, see those mentioned for geographical distribution, notably Ark, 1951; Ark and Thomas, 1946; and Janse, 2006). Furthermore, the bacterium is also known to infect Catasetum spp., Cypripedium spp. (Lady’s slipper), Dendrobium spp., Doritaenopsis hybrids, Epidendrum spp., Epiphronitis veitchii, Ionopsis utricularioides, Miltonia spp., Oncidium spp., Ornithocephalus bicornis, Renanthera spp., Rodri­ cidium spp., Rodriguezia spp., Rhynchostylis spp., Sophronitis carnus, Trichocentrum spp., Vanda spp., Vanilla spp., Vuylstekeara spp. (Janse and Jegen, 1980), and Zygopetalum spp. Remarkably, the parents of Vuylstekeara, a susceptible orchid produced by man and not occurring wild in nature, viz. Cochlioda spp. and Odontoglossum spp., have not been reported as natural hosts of A. cattleyae. Brown spot of orchids and its causal organism have been reported from Australia, New South Wales (Stovold et al., 2001), Belgium, China (Ding et al., 1993), Germany (Weichlein, 2004), Italy (Scortichini et al., 2005), Korea (Han et al., 2009), The Netherlands (Janse, 2006; Janse and Jegen, 1980), The Philippines (Quimio and Tabei, 1979), Poland (Pulawska et al., 2013), Portugal (Martins, 1981), Taiwan (Wey, 1988; Huang, 1990), the United States (Ark, 1951; Ark and Thomas, 1946; Miller, 1990), and probably Venezuela (Trujillo and Hernández, 1999).

A. CATTLEYAE: GENERAL PROPERTIES AND SYMPTOMATOLOGY ■■ Morphological and phenotypic traits of A. cattleyae have been described previously (Pulawska et al., 2013; Schaad et al., 2008; Vaneechoutte et al., 2013; Willems et al., 1992). A. cattleyae is a gram-negative bacterium with rod-shaped, straight to slightly curved cells with dimensions of 0.2–1.2 × 0.8–5.0 µm. Like other Acidovorax spp., it is motile by one polar flagellum. The bacterium can accumulate polyβ-hydroxybutyrate, is oxidase-positive, does not produce levan on 5% sucrose nutrient agar, and does not reduce nitrate. A. cattleyae does not hydrolyze gelatin, but it hydrolyzes starch. The bacterium induces a hypersensitive response (HR) on tobacco (Nico­ tiana tabacum). A. cattleyae can be distinguished from A. avenae, A. citrulli, and A. oryzae by DNADNA reassociation assays, AFLP analysis, and several phenotypic traits (see the following).

FIG. 8.1. Typical symptoms caused by natural infection of orchids by Acidovorax cattleyae. A, Typical, large black leaf spots, surrounded by a light-green or yellow halo, on mature Cattleya orchid. B, Dark-brown leaf spots surrounded by a yellow halo and complete necrosis of leaves on a young Vuylstekeara orchid. (Courtesy CABI—Reproduced, by permission, from Janse, J. D.; 2006; Phytobacteriology: Principles and Practice; CAB International, Wallingford, UK)

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Acidovorax cattleyae: Bacterial Brown Spot of Orchids

Typical symptoms caused by A. cattleyae in several orchid species are shown in Figures 8.1 to 8.3. Small, dark-green, water-soaked spots are the first symptoms caused by A. cattleyae, which can occur on any growth stage of the plant(let). These spots enlarge rapidly under humid conditions and turn brown, then black. These lesions often become surrounded by a yellow halo. In advanced stages of infection, spots are sunken and become invaded by secondary colonizing organisms. Bacterial ooze may drip from leaf lesions, especially where infections reach leaf tips. On Dendrobium, the lesions may also be corky and swollen (oedema), and on Zygopetalum they usually do not develop a yellow halo. Spots may coalesce, and entire leaves and plants may die, especially when the crown and growing point become infected. Lesions can occur on seedlings, on which infection can be lethal. However, infection can also occur on older plants, especially on Cattleya spp., where the disease rarely kills the plant.

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FIG. 8.3. Water-soaked spots in an early stage of disease development, caused by Acidovorax cattleyae on a leaf of Phalaenopsis orchid, following natural infection. (Courtesy J. van Vaerenbergh—Reproduced by permission)

FIG. 8.2. Water-soaked spot, typical of bacterial brown spot infection caused by Acidovorax cattleyae on leaves of Cattleya orchid, following inoculation. A, Water-soaked spots are where cells are leaking due to action of diffusing bacterial toxins, but bacteria are not yet present. B, Yellow, chlorotic ring due to bacterial infection. C, Central necrotic area, where secondary colonizing and saprophytic organisms may occur. (Courtesy CABI—Reproduced, by permission, from Janse, J. D.; 2006; Phytobacteriology: Principles and Practice; CAB International, Wallingford, UK)

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CHAPTER 8

Importantly, some other bacterial pathogens cause diseases of orchids, the symptoms of which can be confused with bacterial brown spot. For instance, Pantoea cypripedii causes small, water-soaked spots on orchid leaves that are often surrounded by yellow halos. The infection by this pathogen spreads rapidly and rots the leaves and roots (in contrast to infections caused by A. cattleyae), but spreads more slowly into the rhizomes or pseudo bulbs. This wet rot may be accompanied by a bad odor (Bradbury, 1977; Janse, 2006). A picture of Paphiopedilum (Cypripedium) maudiae infected with P. cypripedii is shown in Figure 8.4. Burkholderia gladioli pv. gladioli was reported to infect Dendrobium, Oncidium, and Miltonia spp. and related hybrids in orchid nurseries under shade cloth in Hawaii (Keith et al., 2005). Symptoms start as water-soaked spots, but unlike brown spot caused by A. cattleyae, these spots rapidly develop into a gray-brown soft decay of the entire leaf (Keith et al., 2005). Finally, light-brown to black leaf spots that generally do not coalesce, and are often surrounded by yellow halos, were found to be caused by Burk­ holderia andropogonis on Odontoglossum, Odon­ tioda, Odontocidium, and Vuylstekeara spp. in Japan (Takahashi et al., 2004). This disease develops only at relatively low temperatures (<20°C), and infection does not spread to bulbs (Takahashi et al., 2004).

ISOLATION AND DETECTION OF A. CATTLEYAE Isolation A. cattleyae can be readily isolated onto King’s medium B (King et al., 1954) from symptomatic plant tissue as long as the tissue is taken from the margin of healthy and diseased tissue. On King’s medium B, the bacterium produces small (1–3 mm in diameter), nonfluorescent, flat, round, white colonies with regular margins after 3 days of growth at 28°C.

Serology A double antibody sandwich–enzyme linked immunosorbent assay (DAS-ELISA) for A. cattleyae detection in infected plant material is available from Bioreba AG (Reinach, Switzerland). However, the A. cattleyae–specific IgG antibody used in this test cross-reacts with A. citrulli and A. avenae. Nevertheless, according to the European Plant Protection Organisation Standard (EPPO, 2010), this ELISA protocol can be used.

Phenotypic Characteristics

FIG. 8.4. Leaf spots of Paphiopedilum (Cypripedium) maudiae caused by natural infection by the bacterium Pantoea cypripedii, which may be confused with symptoms of bacterial brown spot caused by Acidovorax cattleyae. In the case of P. cypripedii infection, spots often start from the leaf base. (Courtesy CABI— Reproduced, by permission, from Janse, J. D.; 2006; Phytobacteriology: Principles and Practice; CAB International, Wallingford, UK)

A. cattleyae can be distinguished from A. avenae, A. citrulli, and A. oryzae by DNA-DNA reassociation assays, AFLP analysis, and several phenotypic traits. Some phenotypic characteristics that distinguish A. cattleyae from its closest plant-pathogenic relatives are summarized in Table 8.1. A. cattleyae utilizes D -glucose, D -xylose, D -mannitol, D -sorbitol, sodium citrate, adipate, D -mannose, citraconate, D -arabitol, ethanolamine, ethanol, L -arabinose, dulcitol, galactose, glycerol, lactose, and sucrose. The bacterium does not utilize D -fucose. In contrast to A. cattleyae, A. oryzae does not utilize sodium citrate, A. citrulli does not utilize D -mannitol, and neither of these species utilizes D -arabitol. Additionally, A. cattleyae does not utilize maltose, while A. avenae does; and A. cattleyae does not liquefy gelatin, while A. avenae, A. oryzae, and A. citrulli do. To date, A. cattleyae is the only Acidovorax species that does not produce pyrrolidonyl aminopeptidase. Fatty acid analysis can also be used to distinguish A. cattleyae (Stead, 1992), since the bacterium has a distinct fatty acid methyl ester profile (Table 8.2).

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Acidovorax cattleyae: Bacterial Brown Spot of Orchids TABLE 8.1. Phenotypic characteristics useful for distinguishing Acidovorax cattleyae from other phytopathogenic Acidovorax speciesa

Characteristicsb

A. cattleyaec

A. konjaci

A. citrulli

A. avenae

Pyrrolidonyl aminopeptidase

+

+

+

Lactose*

Rhamnose*

(+)

Nitrate reduction

+

+

+

Nitrite reductase

Gelatin hydrolysis

Malonate**

+

Mannitol*

+W

(+)

+

+

(+)

–/(+)

+

Glucose*

–/(+)W

–/(+)W

Xylose*

(+)

–/+W

Arabinose*

+

+

+

Hydrolysis of urea

+

+

+

+

Citrate*

Ethylene glycol*

a Adapted

from Vaneechoutte et al., 2013. Courtesy J. D. Janse—© APS. Acid production on LPPR agar (Laffineur et al. 2002). **, Alkalinization of Simmon’s agar base (Wauters and Vaneechoutte, 2011). c –, Negative; +, positive; (+), delayed positive (2 days); +W, weakly positive; –/+ indicates variability among strains. b *,

TABLE 8.2. Fatty acid methyl ester profiles of Acidovorax cattleyaea

Fatty acid

Systematic name

% present

10:0

Decanoic acid

Trace–0.4

10:0 3OH

3-Hydroxydecanoic acid

3.4–4.3

12:0

Dodecanoic acid

2.3–2.9

12:1 3OH

3-Hydroxydecanoic acid

Trace–1.4

12:0 3OH

3-Hydroxydodecanoic acid

Trace–0.6

14:0

Tetradecanoic acid

1.7–2.1

15:0

Pentadecanoic acid

0.5–0.6

16:1 cis 9

cis-9-Hexadecanoic acid

40.6–41.4

16:0

Hexadecanoic acid

32.0–35.2

17:0 cyclo

cis-9,10-Methylene hexadecanoic acid

0.4–0.5

17:0

Heptadecanoic acid

0.2–0.3

18:1 cis 11

cis-11-Octadecanoic acid

12.7–14.5

18:0

Octadecanoic acid

0.1–0.6

a Adapted

from Gardan et al., 2000; Han et al., 2009; and Stead, 1992. Courtesy J. D. Janse—© APS.

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CHAPTER 8

PCR-Based Assay for Detection and Identification Hseu et al. (2011) developed a conventional PCR assay based on the A. cattleyae–specific primer pair, Ac46f/Ac46r, to detect the pathogen from leaves of infected plants with a sensitivity of 10–100 cells/ ml. Cui Zhen et al. (2010) developed a TaqMan real-time PCR assay based on the ITS sequence of A. cattleyae. This assay facilitated the discrimination of A. cattleyae strains from 28 other bacterial strains belonging to related Acidovorax species. The sensitivity of the test was approximately 9 × 10 –9 µg bacterial genomic DNA.

Molecular Characterization Multilocus sequence analysis (MLSA) using 7 genes (glucose-methanol-choline [gmc]; oxidoreductase [ugpB]; extracellular solute-binding protein, family I; twitching motility protein [pilT]; GTP-binding protein [lepA]; tryptophan synthase subunit beta [trp]; type II citrate synthase [gltA]; and poly(R)-hydroxyalkanoic acid synthase class I [phaC]) has been used to characterize Acidovorax spp. strains according to Giordano (2014). Additionally, whole genome sequencing of the A. cattleyae type strain, DSM17101 (ATCC 33619), is underway at the U.S. Department of Energy Joint Genome Institute (https://gold.jgi.doe.gov/project?id=116390). The 16S rRNA sequence accession for the type strain was deposited as AF137504 (GU339087; 1,491 bp) (Fegan, 2006).

Pathogenicity Tests Pathogenicity tests for A. cattleyae can be performed using a suspension of 106 –107 cells/ml in sterilized 0.01 M phosphate buffered saline (PBS; pH 7.2). Cell suspension of A. cattleyae should be prepared from a pure culture grown for 48 h on nutrient agar or King’s medium B. The cell suspension should be applied with a cotton swab onto leaves slightly injured (punctured) with a sterilized hypodermic syringe. Alternatively, a drop (c. 25 µl) of the cell suspension can be introduced directly into the leaf blade with a syringe. Control plants should be inoculated with sterilized PBS. Inoculated plants should then be maintained under high (>80%) relative humidity, e.g., by covering them with plastic bags for at least 3 days at 24–28°C. As mentioned previously, A. cattleyae

induces a typical hypersensitive response (HR) on tobacco leaves. The HR test can be performed as described by Klement (1963).

EPIDEMIOLOGY AND DISEASE MANAGEMENT ■■ Bacterial brown spot of orchids is a poorly studied disease that has become globally distributed, probably due to the international trade of propagative plant material, including seedlings and breeding material. The origin of the disease and its pathogen is unknown, but could possibly be Southeast Asia (origin of the highly susceptible Phalaenop­ sis and Dendrobium, Paphiopedilum, Renanthera, Rhynchostylis, and Vanda spp.) or South or Central America (origin of the less susceptible Cattleya, and Catasetum, Cochlioda, Epidendrum, Ionopsis, Mil­ tonia, Odontoglossum, Oncidium, Ornithocepha­ lus, Rodriguezia, Sophronitis, Trichocentrum, and Vanilla spp.), where many orchid species have been collected and are cultivated. Many orchids are also exported from these regions as breeding material and seedlings. There have been few thorough epidemiological studies performed on bacterial brown spot of orchids. In 1946, Ark and Thomas observed that the disease was prevalent in greenhouses in California, U.S.A., that maintained high temperatures and high relative humidity (RH) (Ark and Thomas, 1946). As a disease prevention measure, Frank (1988) recommended plant aeration and avoidance of overhead watering. In a study performed at the University of Florida (Tarnowski et al., 2011), the influence of temperature on disease severity was established, with increased brown spot severity correlating with increased temperature (in a range from 15 to 35°C). The influence of RH was explored in a study performed by Ludeking et al. (2011) in Wageningen, the Netherlands, using Phalaenop­ sis plants in greenhouse compartments. This study revealed that when plants were kept at 60–75% RH, disease severity and dispersion of the pathogen was significantly lower than at 90% RH. This information offers a possible bacterial brown spot management strategy while saving energy. Moreover, it was found that treatment of irrigation water with 20 ppm hydrogen peroxide (H 2O2), with a catalyst such as peracetic acid, also substantially reduced the dispersion of A. cattleyae. Under climatological

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Acidovorax cattleyae: Bacterial Brown Spot of Orchids

conditions in Taiwan, it was found that A. cattleyae infections were especially prevalent in spring and autumn, while those of Dickeya chrysanthemi, and possibly Pantoea cypripedii, were more common during the summer (Wey, 1988). Orchid producers apply control measures that involve immediate removal of infected tissue using sterilized knives, followed by a preventative application of a bactericide. Physan 20, a quaternary ammonium chloride, at 19 ml/liter, and Kocide 3000, a copper hydroxide, or Phyton 27, a copper sulfate pentahydrate, both at 150 g/liter of water, are frequently used as preventative bactericides. Copper-containing bactericides should not be used on Dendrobium spp. because of a high risk of phytotoxicity. For effective brown spot epidemic control, bactericides should be applied to infected plants and those in the immediate vicinity, and applications should be repeated weekly. Physan 20 can also be used to decontaminate bench surfaces in the greenhouse. In the past, one part of 8-quinolinol benzoate or one part of the sodium salt of o-hydroxidiphenyl-natriphene to 2,000 parts of water was used to control bacterial brown spot of orchids, as a plant drench (Ark, 1951). From personal experience, plants can be drenched in the above-mentioned solution of 8-quinolinol benzoate, left for an hour, and then rinsed with water to successfully prevent spread of the disease in the greenhouse. A. cattleyae is spread by irrigation water, and warm and moist conditions are conducive for disease development. Therefore, reducing RH and temperature, avoiding overhead watering, and increasing air circulation are recommended to manage bacterial brown spot of orchids.

BASIC ASPECTS OF THE INTERACTION BETWEEN THE PATHOGEN AND ITS HOSTS ■■ Virtually nothing is known about basic aspects (physiological and molecular) of the interaction between A. cattleyae and its orchid hosts. A. cattleyae has been reported to produce antibacterial and antifungal compounds in vitro (Hu and Young, 1998). Interestingly, of all the strains of Acidovorax spp. tested by Hu and Young (1998), only two strains of A. cattleyae inhibited the growth of Listeria innocua. In addition, all A. cattleyae strains tested inhibited the growth of the fungus Rhodotorula mucilaginosa.

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CONCLUSIONS ■■ Bacterial brown spot of orchids, caused by A. cattleyae, is a poorly studied disease of orchid species that has become globally distributed, most probably due to international trade of plant material. The origin of the disease and its pathogen is unknown, but could possibly be Southeast Asia or South/Central America, where many orchid species originated. To date, there is a lack of knowledge about fundamental aspects of plant–pathogen interactions of this disease. In greenhouses, the disease can be effectively managed by reducing temperature and relative humidity, avoiding overhead irrigation, and applying bactericides. This is especially true for the less sensitive orchid species, such as Cattleya. For other orchid species, such as Vuylstekeara and Phalaenopsis, bacterial brown spot management is more challenging, especially in seedlings, and requires early pathogen detection and exclusion. Only a few molecular methods are available for A. cattleyae detection and identification, but recently new PCR-based methods have been developed.

L I T E R AT U R E C I T E D Ark, P. A. 1951. California orchids resistant to some diseases in habitat become susceptible in greenhouse. Calif. Agric. 1951:13-14. Ark, P. A., and Thomas, H. E. 1946. Bacterial leaf spot and bud rot of orchids caused by Phytomonas cattleyae. Phytopathology 36:695-698. Bradbury, J. F. 1977. Erwinia cypripedii. No. 56, Sheet 554, Ref. 4. IMI Descriptions of Fungi and Bacteria. CABI, Wallingford, UK. Cui Zhen, D., Wen Jun, Z., Dong Yi, C., Hong Yun, C., and Shui Fang, Z. 2010. Detection of Acidovorax avenae subsp. cattleyae by real-time fluorescent PCR. Acta Phytopathol. Sin. 40:235-241. Ding, A. D., Liu, C. Z., and Qi, P. K. 1993. Identification of the pathogen causing bacterial brown spot of Phalaenopsis aphrodite Reichb. J. South China Agric. Univ. 14:124-126. EPPO/OEPP. 2010. ELISA tests for plant pathogenic bacteria. EPPO/OEPP Standard PM 7/101/1. Wiley online library: http://onlinelibrary.wiley.com/doi/ 10.1111/j.1365-2338.2010.02420.x/epdf Fegan, M. 2006. Plant pathogenic members of the genera Acidovorax and Herbaspirillum. Pages 671-702 in: Plant-Associated Bacteria. S. S. Gnanamanickam, ed. Springer, Dordrecht, the Netherlands. Frank, D. W. 1988. Control of bacterial soft rot caused by Pseudomonas cattleyae in the culture of Phalaenopsis. Orchid Dig. 52:66-67.

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Gardan, L., Dauga, C., Prior, P., Gillis, M., and Saddler, G. S. 2000. Acidovorax anthurii sp. nov., a new phytopathogenic bacterium which causes bacterial leaf-spot of anthurium. Int. J. Syst. Evol. Microbiol. 50:235-246. Giordano, P. R. 2014. Identification and Characterization of a New Bacterial Disease of Creeping Bentgrass (Agrostis stolonifera L.) Caused by Acidovorax avenae subsp. avenae. Ph.D. thesis. Michigan State University, East Lansing. Han, K. S., Lee, S., Park, J. H., Han, Y. K., Kim, D. H., and Lee, J. S. 2009. Incidence of bacterial brown spot of Phalaenopsis orchids caused by Acidovorax avenae subsp. cattleyae. Res. Plant Dis. 15:183-186. Hseu, S. H., Sung, C. J., and Shentu, H. 2011. A specific primer pair for diagnosis and detection of bacterial brown spot pathogen Acidovorax avenae subsp. cattleyae on Phalaenopsis orchid. Plant Pathol. Bull. 20:52-64. Hu, F. P., and Young, J. M. 1998. Biocidal activity in plant pathogenic Acidovorax, Burkholderia, Herba­ spirillum, Ralstonia and Xanthomonas spp. J. Appl. Microbiol. 84:263-271. Huang, T. C. 1990. Characteristics and control of Pseudo­ monas cattleyae causing brown spot of Phalaenopsis orchid in Taiwan. Plant Prot. Bull. (Taipei, Taiwan) 32:327. Janse, J. D. 2006. Phytobacteriology: Principles and Practice. CABI, Wallingford, UK. Janse, J. D., and Jegen, M. 1980. Vuylstekeara (Cambria), een nieuwe waardplant van Pseudomonas cattleyae. [In Dutch.] Verslagen en Mededelingen Plantenziektenkundige Dienst. 157:25-26. Keith, L. M., Sewake, K. T., and Zee, F. T. 2005. Isolation and characterization of Burkholderia gladioli from orchids in Hawaii. Plant Dis. 89:1273-1278. King, E. O., Ward, M. K., and Raney, D. E. 1954. Two simple media for demonstration of pyocyanin and fluorescein. J. Lab. Clin. Med. 44:301-307. Klement, Z. 1963. Method for rapid detection of the pathogenicity of phytopathogenic Pseudomonas. Nature 199:299-300. Laffineur, K., Janssens, M., Charlier, J., Avesani, V., Wauters, G., and Delmée, M. 2002. Biochemical and susceptibility tests useful for identification of nonfermenting gram-negative rods. J. Clin. Microbiol. 40:1085-1087. Ludeking, D., Hamelink, R., Kromwijk, A., Schenk, M., Vermunt, A., and Woets, F. 2011. Detectie en beheersing van bacterierot veroorzaakt door Pseudomonas cattleyae in Phalaenopsis (Detection and control of bacterial rot caused by Pseudomonas cattleyae in Phalaenopsis). Rep. GTB-1096. [In Dutch with English summary.] Wageningen University, Wageningen, the Netherlands, Martins, J. M. 1981. Bacterial diseases of ornamental plants in Portugal. III. Pseudomonas spp. from orchids. Agron. Lusitania 41:67-75.

Miller, J. W. 1990. Bacterial Brown Spot of Orchid Caused by Pseudomonas cattleyae. Plant Pathol. Circ. 330, Fla. Dept. Agric. Cons. Serv. Division of Plant Industry, Gainesville, FL. Pavarino, G. L. 1911. Malattie causate da bacteri nelle orchidee. Nota preliminare (A bacterial disease of orchids). [In Italian).] Classe di Scienze Fisiche, Matematiche e Naturali 20:233-237. Pulawska, J., Mikicinski, A., and Orlikowski, L. B. 2013. Acidovorax cattleyae—The causal agent of bacterial brown spot of Phalaenopsis lueddemanniana in Poland. J. Plant Pathol. 95:407-410. Quimio, A. J., and Tabei, H. 1979. Identity of the bacterium associated with bacterial brown spot of Phalaenopsis orchids. Philipp. Phytopathol. 15:76-80. Savulescu, T. 1947. Contribution à la classification des bactériacées phytopathogènes. Analele Academiei Romane Ser III, Tom 22, Mem. 4:1-26. Schaad, N. W., Postnikova, E., Sechler, A., Claflin, L. E., Vidaver, A. K., Jones, J. B., Agarkova, I., Ignatov, A., Dickstein, E., and Ramundo, B. A. 2008. Reclassification of subspecies of Acidovorax avenae as A. avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli Schaad et al., 1978) comb. nov., and proposal of A. oryzae sp. nov. Syst. Appl. Microbiol. 31:434-446. Scortichini, M., d’Azenzo, D., and Rossi, M. P. 2005. New record of Acidovorax avenae subsp. cattleyae on orchid in Italy. J. Plant Pathol. 87:244. Stead, D. E. 1992. Grouping of plant-pathogenic and some other Pseudomonas spp. by using cellular fatty acid profiles. Int. J. Syst. Bacteriol. 42:281-295. Stovold, G. E., Bradley, J., and Fahy, P. C. 2001. Acidovorax avenae subsp. cattleyae (Pseudomonas cattleyae) causing leafspot and death of Phalaenopsis orchids in New South Wales. Australas. Plant Pathol. 30:73-74. Takahashi, Y., Takahashi, K., Watanabe, K., and Kawano, T. 2004. Bacterial black spot caused by Burkholderia andropogonis on Odontoglossum and intergeneric hybrid orchids. J. Gen. Plant Pathol. 70:284-287. Tarnowski, T. L., Palmateer, A. J., and McMillan, R. T. 2011. Effect of temperature on bacterial leaf spot of Phalaenopsis, caused by Acidovorax cattleyae. (Abstr.) Phytopathology 101:S175. Trujillo, C., and Hernández, Y. 1999. Bacterial spot in orchid. Fitopatol. Venezuelana 12:5-8. Vaneechoutte, M., Janssens, M., Avesani, V., Delmée, M., and Deschaght, P. 2013. Description of Acidovorax wautersii sp. nov. to accommodate clinical isolates and an environmental isolate, most closely related to Acidovorax avenae. Int. J. Syst. Evol. Microbiol. 63:2203-2206. Wauters, G., and Vaneechoutte, M. 2011. Approaches to the identification of aerobic Gram-negative bacteria. Pages 539-558 in: Manual of Clinical Microbiology, 10th ed. J. Versalovic, K. A. Carroll, G. Funke, J. H. Jorgensen, M. L. Landry, and D. W. Warnock, eds. American Society for Microbiology, Washington, DC.

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Acidovorax cattleyae: Bacterial Brown Spot of Orchids Weichlein, D. 2004. Untersuchungen zur Lebensweise und Bekämpfung von Acidovorax avenae ssp. cattleyae an Phalaenopsis-Hybriden. (Research on the biology and control of Acidovorax avenae subsp. cattleyae in Phalaenopsis-hybrids, in German). Ms.C. thesis. Wiesbaden Technical College, Wiesbaden, Germany. Wey, G. C. 1988. Occurrence and investigation of important diseases on Phalaenopsis in Taiwan. Rep. Taiwan Sugar Res. Inst. 122:31-41.

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Willems, A., Goor, M., Thielemans, S., Gillis, M., Kersters, K., and De Ley, J. 1992. Transfer of several phytopathogenic Pseudomonas species to Acido­ vorax as Acidovorax avenae subsp. avenae subsp. nov., comb. nov., Acidovorax avenae subsp. citrulli, Acidovorax avenae subsp. cattleyae, and Acidovorax konjaci. Int. J. Syst. Bacteriol. 42:107-119.

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