solanacearum race 1, biovar 1 entering Florida in ...

CANADIAN JOURNAL OF PLANT PATHOLOGY 20:171-175, 1998

A distinct pathotype ofRalstonia (Pseudomonas) solanacearum race 1, biovar 1 entering Florida in pothos (Epipremnum aureum) cuttings DJ. Norman and J.M.F. Yuen University of Florida, Plant Pathology Department, Central Florida Research and Education Center, 2807 Binion Rd., Apopka, FL 32703, Florida Agricultural Experiment Station journal series no R-05914. Accepted for publication 1998 04 14 Ralstonia solanacearum was isolated from diseased pothos (Epipremnum aureum) cuttings imported to Florida from Costa Rica. Epipremnum aureum has not previously been described as a host of R. solanacearum. Bacterial isolates were identified by standard bacteriological tests, FAME analysis (MIDI®), metabolic profile (Biolog®), and by PCR using species-specific primers. All isolates were biovar 1, Gram-negative, oxidase negative, catalase positive, motile, strictly aerobic, and accumulated poly-6hydroxybutyrate. Similarity indices using MIDI matched in most cases to R. solanacearum; however, low similarity indices were obtained with the Biolog data base. PCR amplification and electrophoretic analysis revealed a 148 bp product for all pothos isolates, identical to that in R. solanacearum control strains. Pathogenicity of isolates from pothos were compared with tomato and potato isolates from Florida on tomato, potato, pothos, tobacco, and triploid banana. Isolates from pothos caused wilt of tomato and potato, variable symptoms on tobacco, and mild symptoms on banana; they were identified as race 1. Ralstonia solanacearum isolates from pothos represent a distinct pathotype of race 1, biovar 1 and differ from other race 1, biovar 1 strains by being pathogenic on pothos. Norman, D.J., and J.M.F. Yuen. A distinct pathotype of Ralstonia (Pseudomonas) solanacearum race 1, biovar 1 entering Florida in pothos (Epipremnum aureum) cuttings. Can. J. Plant Pathol. 20:171 175. Le Ralstonia solanacearum a ete isole de boutures malades de pothos (Epipremnum aureum) importees en Floride du Costa Rica. L'Epipremnum aureum n'avait pas ete prealablement decrit comme un note du R, solanacearum. Les isolats bacteriens ont ete identifies par des tests bacteriologiques standards, 1'analyse FAME (MIDI®), le profil metabolique (Biolog®) et par la PCR avec des amorces specifiques a Tespece. Tous les isolats appartenaient au biovar 1 et etaient Gram negatif, oxydase negatif, catalase positif, mobiles, strictement aerobics et accumulaient du polyhydroxybutyrate. Les indices de similarite de 1'analyse MIDI correspondaient generalement au R. solanacearum; par contre, de faibles indices de similarite ont ete obtenus avec la base de donnees de Biolog. L'amplification PCR et 1'analyse electrophoretique ont mis en evidence un produit de 148 pb pour tous les isolats du pothos, identique a celui des souches temoins du R. solanacearum. Le pouvoir pathogene des isolats du pothos a ete compare a celui d'isolats floridiens de la tomate et de la pomme de terre sur la tomate, la pomme de terre, le pothos, le tabac et un banane triploide. Les isolats du pothos ont cause du fletrissement sur la tomate et la pomme de terre, des symptomes variables sur le tabac, et des symptomes benlns sur la banane; ils ont ete identifies comme etant de la race 1. Les isolats de R, solanacearum du pothos representent un pathotype distinct de la race 1 biovar 1 et se distinguent des autres souches de la race 1 biovar 1 par leur capacite a affecter le pothos.

The United States tropical foliage industry in 1995 had a wholesale value of over 500 million dollars; with approximately 61% of the production in Florida (Henley 1996). Each year over 680 million dollars worth of nursery stock is imported into the United States, much of which comes from Central and South America (USD A 1996). This article reports the entry of a distinct pathotype of Ralstonia (Pseudomonas) solanacearum E. F. Smith (synonym Burkholderia solanacearum) Race 1 into Florida in pothos (Epipremnum aureum Linden & Andre) cuttings, Ralstonia solanacearum has not been described as a pathogen of pothos or other plants belonging to the family Araceae. Ralstonia solanacearum is a common bacterial pathogen in tropical and subtropical areas of the

extensive host range and are pathogenic on a number of hosts, i.e. tomato, tobacco, ginger, and diploid bananas, whereas the other four races have more limited host ranges, with Race 2 infecting Heliconia and triploid banana, Race 3 infecting potato, Race 4 infecting mulberry, and Race 5 infecting ginger. Hay ward (1964) developed the biovar-based classification system that differentiated strains into four biovars based on their ability to utilize maltose, lactose, cellobiose, mannitol, sorbitol, and dulcitol. He et al. (1983) later added a fifth utilization pattern. Very little correlation exists between the race and biovar classification systems. More recently others have also begun to identify and classify this heterogeneous pathogen using restriction fragment-length polymorphisms (RFLP) analysis (Cook et al. 1989,

world and is known to infect hundreds of plant

Gillings & Fahy 1993), fatty acid methyl ester

species comprising 44 families (Hayward 1991). Ralstonia solanacearum has been classified into five races and five biovars based on host range and carbon source utilization, respectively. Race 1 strains have an

(FAME) analysis (Janse 1991), DNA homology (Palleroni & Doudoroff 1971), repetitive sequence (rep) - PCR (Smith et al. 1995), and the use of tRNA consensus primers (Seal et al. 1992a). 171

172 CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 20, 1998

In the summer of 1996, disease symptoms were observed on single-node cuttings of pothos 'Marble Queen' that had been imported from Costa Rica. Primary symptoms were chlorosis and dark discolored leaf veins (Fig. 1). Two weeks after planting cuttings into synthetic potting mix, no roots had developed and stems had begun to decay from the cut base upward. Bacterial ooze exuded from discolored vascular bundles in cut ends of infected stems. Bacteria were isolated from surface sterilized (0.3% NaOCl) leaves and stems of infected cuttings on modified Kelman's (Norman & Alvarez 1989) triphenyltetrazolium chloride medium (TZC). Individual colonies were selected and assessed for purity after incubation at 27 ± 1°C for 48 h. Selected colonies were mucoid and displayed the characteristic red swirling egg-shaped pigmentation pattern of R. solanacearum (Kelman 1954). Forty isolates were collected over an 8-month period from nurseries in Lake and Orange counties in Florida. Isolates were recovered from both 'Marble Queen' and 'Golden' pothos cuttings that had been recently imported from Costa Rica. At two nurseries, strains were also isolated from large stock plants originally obtained as cuttings from Costa Rica in the previous year. Individual vines in these stock plants displayed symptoms of chlorosis and dark vein necrosis; single node cuttings taken from these plants did not root and eventually died. All pothos isolates were characterized on yeast dextrose calcium carbonate medium (YDC) (Wilson et al. 1967) for colony color and morphology, King's Media B (KMB) for fluorescence (King et al. 1954) and on Nile Blue Medium (NBA) for accumulation of poly-B-hydroxybutyrate (Pierce & Schroth 1994). They grew readily on YDC medium and formed large, round, mucoid, beige colonies within 48 h. None of the strains fluoresced under long-wave ultraviolet light on KMB; however, all fluoresced orange on NBA medium, indicating poly-B-hydroxybutyrate formation. All pothos isolates tested in this study were Gram-negative, oxidase negative, catalase positive, motile, and strictly aerobic on the basis of tests conducted following protocols of Suslow et al. (1982) and MacFaddin (1981). Fatty acid (FAME) analyses were performed on the 40 bacterial isolates using the MIDI Microbial Identification System, software version TSBA 3.90 (Microbial ID, Inc., Newark, DE) according to standard procedures (Miller 1982, Sasser 1990a, 1990b). All isolates from pothos were identified as R. solanacearum with a mean similarity index (si) of 0.894. Mean si from multiple fatty acid extracts of control strains to correct species identification were as follows: Stenotrophomonas maltophilia 0.836; Xanthomonas campestris pv. begoniae 0.863; R.

solanacearum ATCC control strains 33192 and 33193, 0.871 and 0.860 respectively; and for Florida tomato (7 strains) and potato (8 strains) respectively 0.654 and 0.506. Tomato and potato strains (race 1, biovar 1) of R. solanacearum had been isolated from various locations in Florida (strains provided by J. B. Jones, D. O. Chellemi and D. P. Weingartner, University of Florida; and T. S. Shubert, Division of Plant Industry, Florida). Similarity indices for both FAME and Biolog analysis > 0.50 were considered matches with 1.0 considered a perfect match. Pothos strains were classified, based on their metabolic profile using the 95 carbon-source Biolog GN System (Biolog, 3938 Trust Way, Hayward CA) and accompanying software (Version 3.5). Using standard procedures, 35% (14/40) of the isolates were identified as R. solanacearum (si = 0.361). The other 26 isolates had similarities to other genera with si < 0.5. Average si values for control strains were as follows: tomato strains, 0.457; potato strains, 0.641; and ATCC cultures 33192 and 33193, 0.769 and 0.357, respectively. Ralstonia solanacearum species-specific primers (PS96-H, PS96-I) and protocols developed by Seal et al. (1992b) were utilized in a polymerase chain reaction (PCR) on all pothos and R. solanacearum control strains indicated above. Erwinia herbicola (E101) was utilized as a negative control in each amplification. Amplifications were performed using a PTC100HB thermal cycler (MJ Research, Inc.). A PCR product of 148 bp was detected after PCR amplification and electrophoretic analysis of all pothos and R. solanacearum control strains, in agreement with Seal et al. (1992b). No bands were observed with the E. herbicola control strain. These results confirmed the identity of the pothos strains as R. solanacearum. Biovar determination of bacterial isolates from pothos were performed following the procedures of Hayward (1964). None of the bacterial isolates from pothos were able to utilize the six carbon sources used for biovar differentiation and therefore were classified as biovar 1. All 40 pothos isolates were tested for pathogencity on tomato (cv. Walter), potato (cv. Red Pontiac), pothos (cv. Golden), tobacco (cv. Hicks) and triploid banana (cv. Dwarf Cavendish). Tomato plants were inoculated by pouring 10 mL of a 24-h-old aqueous suspension of bacteria spectrophotometrically adjusted (A600) to 1 x 108 cfu/mL, onto the soil surface of three 10-cm pots (450 cm 3 ) each containing one tomato plant. All R, solanacearum isolates from pothos, tomato, and potato caused wilt symptoms and plant death when inoculated onto tomato. First signs of wilt began 7 days after application of inoculum. For pothos inoculations, 10 single-node cuttings were placed into 20 mL of a bacterial suspension prepared

NORMAN, YUEN: POTHOS/RALSTONIA

173

Figure 1. A) Symptoms of bacterial wilt on Epipremnum aureum; B) Characteristic dark necrotic veins on a leaf, caused by Ralstonia solanacearum.

174

CANADIAN JOURNAL OF PLANT PATHOLOGY, VOLUME 20, 1998

in the same way, for 15 min, before planting into 15cm pots (1539 cm3) at five cuttings per pot. Plants were placed in a glasshouse with temperatures maintained between 18 and 32°C and maximum lighting at 266 Limol-irr^s'1 and examined over a 4-week period for symptom development. All pothos cuttings inoculated with R. solanacearum isolated from pothos developed symptoms within 1 week. The first symptom was chlorosis followed by dark vein necrosis, wilting, and finally plant death. Within 3 weeks, 69.5% (278/400) of the cuttings inoculated with the 40 isolates were dead. The remaining 30.5% of the cuttings exhibited wilt, chlorosis, and leaf necrosis, but these symptoms were not restricted to any one pothos isolate. Ralstonia solanacearum was easily reisolated from symptomatic plants onto TZC medium. When pothos cuttings were inoculated with R. solanacearum strains isolated from tomatoes or potatoes, cuttings rooted normally and did not develop disease symptoms. Banana and potato plants were inoculated with 24h-old cultures of each of the pothos strains using a needle dipped in a bacterial suspension then stabbed into the stems and pseudo-stems of three 30-cm-tall potato and banana plants, respectively. Plants were observed over a 30-day period for symptom development. Eighty percent (32/40) of the pothos strains produced mild disease symptoms on banana. Symptoms included dark veins, leaf necrosis and chlorosis; however, wilt and death of the plants did not occur. After initial symptom development, banana plants produced leaves without further disease symptoms. However, R. solanacearum was consistently isolated from the vascular tissue of banana plants that had developed symptoms initially. One of the R. solanacearum control strains from tomato also produced mild symptoms on banana. No symptoms developed on banana when the other 14 strains from tomato and potato were inoculated onto banana (Table 1). All R. solanacearum isolates from pothos, tomato, and potato caused wilt symptoms and plant death when inoculated onto potato. Bacteria were

reisolated and characterized, as previously described, from representatives of each replicate group of plants. In all inoculation experiments three controls were used: 1) SDW, 2) E. herbicola (strain E101), and 3) the R. solanacearum control strains from potato and tomato. The ability of the pothos strains to induce hypersensitive reaction (HR) on tobacco Hicks was tested as described by Lozano and Sequeira (1970) on at least two leaves of individual tobacco plants. Observations of HR response was done at 12, 24, and 36 h. Plants were maintained in the greenhouse for 4 weeks to monitor for evidence of infection. All isolates from pothos caused leaf mesophyll collapse (HR) within 12 to 36 h on tobacco. Seven of the isolates also produced symptoms of wilt, chlorosis, or vein necrosis over a 4-week period. Ralstonia solanacearum was first identified on tomato in Florida in 1897 (Rolf 1898). Studies have classified strains infecting eggplant, potato, tomato, and sunflower in Florida as race 1, biovar 1 (Martin et al. 1982, McLaughlin & Sequeira 1989, Velupillai & Stall 1984). There are no records of other races or biovars affecting crop production in Florida. The R. solanacearum strains from pothos represent a distinct pathotype of race 1, biovar 1, which differ from Florida strains by being readily capable of infecting, limiting growth, and causing death of pothos. Positive identification to species was obtained by FAME analysis and DNA amplification using species-specific primers. Metabolic profiles (Biolog) were less conclusive because of low similarity indices. We attribute this to these being very fastidious strains that require incubation periods longer then 48 h for carbon source utilization. Black and Sweetmore (1992), using the 95-substrate Biolog system, examined the metabolic profiles of 64 isolates of R. solanacearum from different hosts and geographical areas. Metabolic profiles produced in their study differ from profiles produced by our pothos isolates. The former showed > 92% utilization of a-D glucose, sucrose, c/s-aconitic acid, and L-

Table 1. Host range of R. solanacearum isolates recovered from imported pothos compared to those from tomato and potato plants indigenous to Florida Percentage with symptoms Source of R. solanacearum isolates0 Bioassay host species

Cultivar

Pothos (Epipremnum aureum Linden & Andre) Tomato (Lycopersicon esculentum Mill) Potato (Solanum tuberosum L.) Banana (Musa acuminate L.)

Golden Pothos Walter Red Pontiac Dwarf Cavendish

aAll

Pothos 100a

100 100 80b

isolates produced wilt, chlorosis, and necrosis of plants. bPercent of isolates producing symptoms of vein necrosis and chlorosis without wilt. cNumber of isolates tested from pothos, tomato and potato were 40, 8, and 7, respectively.

Tomato

0 100 100 12.5b

Potato

0 100 100 0

NORMAN, YUEN: POTHOS/RALSTONIA 175

threonine, while < 3% of our pothos isolates utilized a-D glucose, sucrose, cw-aconitic acid, and none utilized L-threonine. In this study, growth of pothos isolates did occur in O/F glucose tubes under aerobic conditions; however, basic reactions indicated utilization of tryptone as opposed to glucose. The heterogenic nature of R. solanacearum and its world-wide distribution has enabled this pathogen to develop an extensive host range. Since this pathogen has a history of being transported in propagative plant material (Buddenhagen 1961, Thurston 1963, Harrison 1961), the potential movement of R. solanacearum and other systemic pathogens between countries has probably been greatly underestimated. Ralstonia solanacearum has not been described previously as a pathogen of pothos, a native of Southeast Asia (Bailey & Bailey 1976). In recent years pothos has been extensively cultivated in Costa Rica for the commercial horticulture industry. Plants are grown in mounds, in areas cleared of thick undergrowth. Water movement through these plantings is unchecked and weeds or native plants, commonly associated with the pothos crop, could have served as a source of inoculum from which a new pathotype developed. Bailey, L.H., and E.Z. Bailey. 1976. Hortus Third. Liberty Hyde Bailey Hortorium. Black, R., and A. Sweetmore. 1992. Identification and characterization of Pseudomonas solanacearum using metabolic profiles. Pages 32-44 in G.L. Harman and A.C. Hayward, eds., Bacterial Wilt. Proc. Int. Conf. Kaohsiung, Taiwan. Buddenhagen, I.W. 1961. Bacterial wilt of bananas: History and known distribution. Trop. Agric. (Trinidad) 38:107-121. Cook, D., E. Barlow, and L. Sequeira. 1989. Genetic diversity of Pseudomonas solanacearum: Detection of restriction fragment length polymorphisms with DNA probes that specify virulence and hypersensitive response. Mol. Plant-Microbe Interact, 2:113-121. Gillings, M., and P. Fahy. 1993. Genetic diversity of Pseudomonas solanacearum biovars 2 and N2 assessed using restriction endonuclease analysis of total genomic DNA. Plant Pathol. 42:744-753. Harrison, D.E. 1961. Bacterial wilt of potatoes, field symptoms of the disease and studies on the causal organism, Pseudomonas solanacearum variety asiaticum. Aust. J. Agric. Res. 12: 854-871. Hayward, A.C. 1964. Characteristics of Pseudomonas solanacearum. J. Appl. Bacteriol. 27:265-277. Hayward, A.C. 1991. Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu. Rev. Phytopathol. 29:65-87. He, L.Y., L. Sequeira, and A. Kelman. 1983. Characteristics of strains of Pseudomonas solanacearum from China. Plant Dis. 67:1357-1361. Henley, D. 1996. A revival in foliage. Greenhouse Grower 14:37-38. Janse, J.D. 1991. Infra- and intraspecific classification of Pseudomonas solanacearum strains, using whole cell fatty acid analysis. Syst. Appl. Microbiol. 14:335-345. Kelman, A. 1954. The relationship of pathogenicity in Pseudo-

monas solanacearum to colony appearance on tetrazolium medium. Phytopathology 44:693-695. King, E.G., M.K. Ward, and D.E. Raney. 1954. Two simple media for the demonstration of phyocyanin and fluorescein. J. Lab. Clin. Med. 44:301-307. Lozano, J.C., and L. Sequeira. 1970. Differentiation of races of Pseudomonas solanacearum by leaf infiltration technique. Phytopathology 60:833-838. MacFaddin, J.F. 1981. Biochemical Tests for Identification of Medical Bacteria. Second Edition. Williams and Wilkins, Baltimore / London, pp. 527. McLaughlin, R.J., and L. Sequeira. 1989. Phenotypic diversity in strains of Pseudomonas solanacearum isolated from a single potato field in Northeastern Florida. Plant Dis. 73:960-964. Martin, C., E.R. French, and U. Nydegger. 1982. Strains of Pseudomonas solanacearum affecting solanaceae in the Americas. Plant Dis. 66:458-460. Miller, L.T. 1982. Single derivation method for routine analysis of bacterial whole-cell fatty acid methyl esters, including hydroxy acids. J. Clin. Microbiol. 16:584-586. Norman, D., and A. Alvarez. 1989. A rapid method for the presumptive identification of Xanthomonas campestris pv. dieffenbachiae and other xanthomonads. Plant Dis. 73:654-658. Palleroni, N.J., and M. Doudoroff. 1971. Phenotypic characterization and deoxyribonucleic acid homologies of Pseudomonas solanacearum, J. Bacteriol. 107:690-696. Pierce, L., and M.N. Schroth. 1994. Detection of Pseudomonas colonies that accumulate poly-B-hydroxybutyrate on Nile Blue Medium. Plant Dis. 78:683-685. Rolf, P.H. 1898. Diseases of tomato. Fla. Agric. Expt. Stn. Bull. 47:128-136. Sasser, M.J. 1990a. Identification of bacteria through fatty acid analysis. Pages 199-204 in Z. Klement, K. Rudolph, and D. Sands, eds., Methods in Phytobacteriology. Akademiai Kiado, Budapest. Sasser, M.J. 1990b. Technical note #101: Identification of bacteria by gas chromatography of cellular fatty acids. MIDI, 115 Barksdale Professional Center, Newark, DE 19711. Seal, S.E., L.A. Jackson, and M.J. Daniels. 1992a. Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification. Appl. Environ. Microbiol. 58:3759-3761. Seal, S.E., L.A. Jackson, and M.J. Daniels. 1992b. Isolation of a Pseudomonas solanacearum-specific DNA probe by subtraction hybridization and construction of species-specific oligonucleotide primers for sensitive detection by polymerase chain reaction. Appl. Environ. Microbiol. 58:3751-3758. Smith, J.J., L.C. Offord, M. Holderness, and G.S. Saddler. 1995. Genetic diversity of Burkholderia solanacearum (synonym Pseudomonas solanacearum} race 3 in Kenya. Appl. Environ. Microbiol. 61:4263-4268. Suslow, T.V., M.N. Schroth, and M. Isaka. 1982. Application of a rapid method for gram-differentiation of plant pathogenic and saprophytic bacteria without staining. Phytopathology 72:917-918. Thurston, H.D. 1963. Bacterial wilt of potatoes in Colombia. Am. Potato 1.40:381-390. United States Department of Agriculture (USDA). 1996. Agricultural Statistics, 1995-1996. National Agricultural Statistics Service. Velupillai, M., and R.E. Stall. 1984. Variation among strains of Pseudomonas solanacearum from Florida. Proc. Fla. State. Hort. Soc. 97:209-213. Wilson, E.E., F.M. Zeitoun, and D.L. Fredrickson. 1967. Bacterial phloem canker, a new disease of Persian walnut trees. Phytopathology 57:618-621.