Bacterial Stem Rot of Poinsettia Caused by a Dickeya sp. (Pectobacterium chrysanthemi) in China

In December 2006, a rot symptom of unknown etiology was observed on stems of plants (Euphorbia pulcherrima cv. Fu-xing) at a flower nursery in the Zhejiang Province of China where we had previously reported leaf spot of poinsettia caused by Xanthomonas campestris (2). Chlorotic spots anywhere along the stem and purplish black petioles were the first noticeable symptoms. The spots rapidly coalesced, forming large irregular chlorotic areas. Petioles turned black and shriveled and affected leaves wilted. Infected tissues were soft and water soaked. Ten bacterial strains were isolated from the diseased samples and five were selected for identification. They were similar to those of the standard reference strains of Pectobacterium chrysanthemi (Dickeya sp.), LMG 2804 from Belgium and ZUPB20056 from China, in phenotypic tests based on the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatography of fatty acid methyl esters (FAME) using the Microbial Identification System (MIDI Inc, Newark, DE) with aerobic bacterial library (TABA50), and transmission electron microscopy (TEM,KYKY-1000B, Japan). All strains tested were gram-negative facultative anaerobic rods measuring 1.5 to 3.6 × 0.6 to 1.1 μm, with peritrichous flagella. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They were negative for trehalose and positive for phosphatase production and reducing substances from sucrose. A hypersensitive reaction was observed on tobacco cv. Benshi, 24 h after inoculation. All five isolates, LMG 2804, and ZUPB20056 were identified as P. chrysanthemi (Dickeya sp.) with a Biolog similarity index of 0.58 to 0.83, 0.68, and 0.72 and a FAME similarity index of 0.52 to 0.80, 0.59, and 0.70, respectively. Identification as P. chrysanthemi (Dickeya sp.) was confirmed by PCR with specific primers used by Nassar et al (3). Koch's postulates were completed with the inoculation of 12 4-month-old intact poinsettia plants of cv. Fu-xing with cell suspensions containing 108 CFU/ml by a pinprick at the base of the stem. All five strains induced stem infection similar to those observed in natural infections. No symptoms were noted on the two control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of poinsettia plants. P. chrysanthemi (Dickeya sp.) was first reported in United States as the cause of bacterial stem rot of poinsettia in 1972 (1). To our knowledge, this is the first report of poinsettia stem rot caused by P. chrysanthemi (Dickeya sp.) in China. The disease cycle and the control strategies of the bacterial stem rot of poinsettia in the regions are being further studied. References: (1) H. A. J. Hoitink et al. Plant Dis. Rep. 56:480, 1972. (2) B. Li et al. Plant Pathol. 55:293, 2006. (3) A. A. Nassar et al. Appl. Environ. Microbiol. 62:2228, 1996.

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Bacterial Stem Rot of Oncidium Orchid Caused by a Dickeya sp. (ex Pectobacterium chrysanthemi) in Mainland China

Plant Disease ◽

10.1094/pdis-93-5-0552b ◽

2009 ◽

Vol 93 (5) ◽

pp. 552-552 ◽

Cited By ~ 2

Author(s):

B. Li ◽

W. Qiu ◽

Y. Fang ◽

G. L. Xie

Keyword(s):

Mainland China ◽

Stem Rot ◽

Bacterial Suspension ◽

Identification System ◽

Microbial Identification ◽

Embl Accession ◽

Initial Symptoms ◽

Leaf Tissues ◽

Pectobacterium Chrysanthemi ◽

Microbial Identification System

In December 2007, stem rot symptoms on orchids (Oncidium Gower Ramsey) were observed at a flower nursery in the Zhejiang Province of China. Initial symptoms were water-soaked lesions starting at the base of the stem. As these lesions expanded and elongated, the stem and leaf tissues became soft and watery. When examined with a microscope, cut edges of symptomatic stem and leaf tissues consistently exhibited bacterial streaming. The bacteria were isolated by streaking on nutrient agar (3). All isolates were gram-negative, facultative, anaerobic rods with peritrichous flagella. Infiltration of tobacco leaves (Nicotiana tabacum cv. Samsun) with the bacterial suspension of 108 CFU/ml resulted in typical hypersensitivity reactions within 24 h. Five representative isolates were further characterized by the Biolog Microbial Identification System, version 4.2 (Biolog Inc., Hayward, CA) and gas chromatography of fatty acid methyl esters (FAME) by the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TSBA50). The five isolates were identified as Erwinia chrysanthemi (Pectobacterium chrysanthemi) with a Biolog and FAME similarity index of 0.81 to 0.88 and 0.62 to 0.75, respectively. The transfer of P. chrysamthemi to a novel genus, Dickeya gen. nov., was recently proposed (2). The almost complete 16S rDNA sequence from Oncidium isolate SCH-01 (1,604 bp; EMBL Accession No. FM946179) was determined according to the method of Li et al. (1). A subsequent GenBank search showed that this isolate is 98% identical to that of type strain CFBP 1269T of Dickeya dadantii (EMBL Accession No. AF520707) and CFBP 1200T of Dickeya dianthicola (EMBL Accession No. AF520708). Nevertheless, species identification within genus Dickeya is still difficult since only a limited number of strains of each species have been characterized fully. Koch's postulates were completed with the inoculation of Oncidium seedlings with cell suspensions (108 CFU/ml) by a pinprick at the base of the stem. All five representative isolates induced stem rot similar to that observed in natural infections. No symptoms were noted on the control plants inoculated with sterilized distilled water by the same method. The bacterium was reisolated from symptomatic stems of Oncidium plants. To our knowledge, this is the first report of stem rot on Oncidium orchid in Mainland China caused by the bacterium formerly referred to as P. chrysanthemi, now proposed as Dickeya sp. References: (1) B. Li et al. J. Phytopathol. 154:711, 2006. (2) R. Samson et al. Int. J. Syst. Evol. Microbiol. 55:1415, 2005. (3) N. W. Schaad et al. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society. St. Paul, MN, 2001.

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Bacterial Wilt of Mulberry (Morus alba) Caused by Enterobacter cloacae in China

Plant Disease ◽

10.1094/pdis-92-3-0483b ◽

2008 ◽

Vol 92 (3) ◽

pp. 483-483 ◽

Cited By ~ 14

Author(s):

G. F. Wang ◽

K. Praphat ◽

G. L. Xie ◽

B. Zhu ◽

B. Li ◽

...

Keyword(s):

Bacterial Wilt ◽

Methyl Esters ◽

Enterobacter Cloacae ◽

The United States ◽

Hypersensitive Reaction ◽

Identification System ◽

Bacterial Disease ◽

Bacterial Strains ◽

Microbial Identification ◽

Light Yellow

In August of 2006, a new bacterial disease was noted in Hangzhou mulberry orchards of Zhejiang Province, China where bacterial wilt of mulberry caused by Ralstonia solanacearum was previously reported (3). In the summer, the disease caused severe wilt, especially on 1- or 2-year-old mulberry plants, that resulted in premature plant death. Leaf wilt symptoms generally started on older leaves at the bottom of the plant and spread to the younger leaves. The leaves of infected plants became withered and dry, turned dark brown, and eventually the plants became defoliated. The root xylem of infected plants was moist and discolored with brown stripes. The phloem was asymptomatic, however, in severe infections, the phloem was decayed. The observation of wilting proceeding from the bottom of the plant to the top distinguishes this disease from bacterial wilt caused by R. solanacearum. Five bacterial strains isolated from infected mulberry plants showed characteristics similar to those of the standard reference strain of Enterobacter cloacae subsp. cloacae IBJ0611from China, but differed from R. solanacearum IBJ35, E. cancerogenus LMG2693T, and E. cloacae subsp. dissolvens LMG2683T from the University of Gent, Belgium in phenotypic tests, including the Biolog Identification System version 4.2 (Biolog Inc., Hayward CA), pathogenicity tests, transmission electron microscopy (TEM,KYKY-1000B, Japan) observation, and gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Company, Newark, DE) with the aerobic bacterial library (TABA50). Isolates were gram negative, facultative anaerobic, rod shaped, 0.3 to 1.0 × 1.0 to 3.0 μm with peritrichous flagella. Colonies on nutrient agar were light yellow, smooth, circular, entire, and convex with no green fluorescent diffusible pigment on King's medium B (3). Weak hypersensitive reaction was observed on tobacco 3 days after inoculation. All five strains were identified as E. cloacae with Biolog similarity of 0.662 to 0.863 and FAMEs similarity of 0.632 to 0.701. Inoculation of 10 6-month-old intact mulberry plants of cv Husang with cell suspensions containing 109 CFU/ml by pinprick at the base of the stem reproduced symptoms observed in natural infections. No symptoms were noted on the two control plants inoculated by the same method but with sterilized distilled water. The bacterium was reisolated from the symptomatic mulberry plants. E. cloacae has been reported from the United States as the cause of internal yellowing of papaya fruits (1) and rhizome rot of edible ginger (2). To our knowledge, this is the first report of mulberry wilt caused by E. cloacae in China. References: (1) K. Nishijima et al. Plant Dis. 71:1029, 1987. (2) K. Nishijima et al. Plant Dis. 88:1318, 2004. (3) L. Xu et al. Acta Phytophylacica. Sin. 34:141, 2007.

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Wildfire of Soybean Caused by Pseudomonas syringae pv. tabaci, a New Disease in Korea

Plant Disease ◽

10.1094/pdis-93-11-1214a ◽

2009 ◽

Vol 93 (11) ◽

pp. 1214-1214 ◽

Cited By ~ 3

Author(s):

I.-S. Myung ◽

J.-W. Kim ◽

S. H. An ◽

J. H. Lee ◽

S. K. Kim ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Pathogenic Bacteria ◽

Similarity Index ◽

Soft Rot ◽

Universal Primers ◽

Identification System ◽

Bacterial Disease ◽

Bacterial Strains ◽

Microbial Identification ◽

Soybean Leaves

In 2006 and 2007, a new bacterial disease was observed in field-cultivated soybeans in Boeun District and Munkyung City of Korea. The disease caused severe blighting of soybean (Glycine max) leaves. Soybean leaves in fields showed yellowish spots with brown centers. Brown and dead areas of variable size and shape were surrounded by wide, yellow haloes with distinct margins. Spots might coalesce and affected leaves fell readily. Seven bacterial strains were isolated from chlorotic areas of soybean leaves and all produced white colonies on trypticase soy agar. With the Biolog Microbial Identification System, version 4.2, (Biolog Inc., Hayward, CA) all strains and Pseudomonas syringae pv. tabaci CFBP2106T were identified as P. syringae pv. tabaci with a Biolog similarity index of 0.28 to 0.52 and 0.48 after 24 h. Pathogenicity of the strains (three plants per strain) on soybean leaves at the V5 stage (cv. Hwanggeum) was confirmed by rub inoculation with bacterial suspensions (1 × 108 CFU/ml) in sterile distilled water on the lesions cut 1 cm long on the upper side of the leaves with razor blades and by pinprick on 3-week-old leaves of tobacco (Nicotiana tabacum cv. Samsun) in the greenhouse. Wildfire symptoms on the soybean leaves and faint halos on tobacco leaves were observed 4 days after inoculation. The identification of reisolated bacterial strains was confirmed with the metabolic fingerprintings on Biolog. LOPAT tests (1) and phenotypic characteristics (3) of the strains were similar to those of the CFBP2106T. Colonies were levan positive, oxidase negative, potato soft rot negative, arginine dihydrase negative, and tobacco hypersensitivity negative. All strains were gram-negative, aerobic rods with a polar flagellum. Strains were negative for esculin hydrolysis, gelatin liquefaction, urea production, accumulation of poly-β-hydroxy butyrate, starch hydrolysis, ornithine dihydrolase, lysine dihydrolase, growth at 37°C, utilization of geraniol, benzoate, cellobiose, sorbitol, trehalose, l-rhamnose, and adonitol. Positive reactions were catalase and arbutin hydrolysis, utilization of sorbitol, d-arabinose, and dl-serine. The strains were variable in utilization of mannitol, sucrose, and d-arabinose. The 1,472-bp PCR fragments of strains, BC2366 (GenBank Accession No. FJ755788) and BC2367 (No. FJ755789) was sequenced using 16S rDNA universal primers (2). The sequences shared 100% identity with the analogous sequences of P. syringae pv. glycenea (GenBank Accession No. AB001443) available in NCBI databases. Based on the phenotypic, genetic, and pathological characteristics, all strains were identified as P. syringae pv. tabaci. To our knowledge, this is the first report of P. syringae pv. tabaci causing wildfire on soybean in Korea. References: (1) R. A. Lelliott et al. J. Appl. Bacteriol. 29:470, 1966. (2) I.-S. Myung et al. Plant Dis. 92:1472, 2008. (3) N. W. Schaad et al., eds. Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. The American Phytopathological Society, St. Paul, MN, 2001.

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First Report of Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China

Plant Disease ◽

10.1094/pdis-92-5-0832c ◽

2008 ◽

Vol 92 (5) ◽

pp. 832-832 ◽

Cited By ~ 2

Author(s):

L. H. Xu ◽

G. L. Xie ◽

B. Li ◽

B. Zhu ◽

F. S. Xu ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Similarity Index ◽

Nutrient Agar ◽

Identification System ◽

Pyrus Pyrifolia ◽

Rrna Gene ◽

Bacterial Disease ◽

First Report ◽

Microbial Identification ◽

Microbial Identification System

In the spring of 2006, a new bacterial disease was noted in pear orchards near Hangzhou, Zhejiang Province, China. The disease caused severe blossom blast on pears (Pyrus pyrifolia; cv. Cuiguan). Early symptoms of the disease included blackening of the calyx end of developing fruit, blackening of blossom clusters while leaves of affected blossom clusters appeared normal, or death of clusters consisting of both blossoms and leaves. Later, tips of twigs turned dark brown and died. No bacterial ooze was observed. Twelve bacterial isolates were recovered from ten samples of buds and blossoms. Six isolates were selected for identification. They were similar to those of the reference strains of Pseudomonas syringae pv. syringae LMG5570 and LMG 2230 from Belgium in phenotypic tests on the basis of the Biolog Microbial Identification System (version 4.2; Biolog Inc., Hayward, CA), pathogenicity tests, gas chromatographic analysis of fatty acid methyl esters (FAMEs) using the Microbial Identification System (MIDI Inc., Newark, DE) with aerobic bacterial library (TABA50), and electron microscopy (TEM, KYKY-1000B, Japan). All isolates tested were gram-negative, aerobic rods measuring 1.5 to 2.4 × 0.5 to 0.6 μm with 2 to 4 polar flagella. Fluorescent green diffusible pigment was produced on King's Medium B. Colonies were gray-white and slightly raised with smooth margins on nutrient agar. They produced levan on sucrose nutrient agar. A hypersensitive reaction was observed on tobacco cv. Benshi 24 h after inoculation. All isolates were identified as P. syringae pv. syringae with Biolog similarity index of 0.57 to 0.86 and FAME similarity index of 0.58 to 0.81. Identification as P. syringae pv. syringae was confirmed using 16S rDNA universal primers (2,3): 5′-AGA GTT TGA TCA TGG CTC AG-3′ forward primer, 5′-ACG GTT ACC TTG TTA CGA CTT-3′ reverse primer. The PCR fragments of the three isolates were sequenced and compared with sequences in GenBank. They had 99% similiarity with P. syringae pv. syringae 16S rRNA gene strain NCPPB 3869. Koch's postulates were conducted on buds of the original pear cultivar growing in pots and detached pear blossoms in flasks by spray inoculation with cell suspensions containing 108 CFU/ml of the six isolates at 18 to 22°C with two replications. The bacteria induced symptoms on buds and blossoms similar to those observed in the field. The bacterium was reisolated from symptomatic pear buds and internal ovary tissues. P. syringae pv. syringae was first reported in England as the cause of pear blossom blast in 1914 (1). After searching all the Chinese agricultural databases and major journals (National Knowledge Infrastructure database, Vip Chinese periodical database, Chinese wanfang database, China InfoBank, Scientia Agricultura Sinica, Acta Phytopathologica Sinica, Acta Phytophylacica Sinica, and Journal of Fruit Science), to our knowledge, this is the first report of pear blossom blast caused by P. syringae pv. syringae in China. The disease cycle on pear trees and the control strategies in the regions are being further studied. References: (1) B. P. Barker et al. Ann. Appl. Biol. 1:85, 1914. (2) U. Edward et al. Nucleic Acids Res. 17:7843,1989. (3) B. Li et al. J. Phytopathol. 34:141, 2006.

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

Plant Disease ◽

10.1094/pdis.1999.83.3.300a ◽

1999 ◽

Vol 83 (3) ◽

pp. 300-300 ◽

Cited By ~ 8

Author(s):

D. J. Norman ◽

J. M. F. Yuen

Keyword(s):

Xanthomonas Campestris ◽

Polymerase Chain Reaction Amplification ◽

Methyl Esters ◽

Electrophoretic Analysis ◽

Identification System ◽

Strictly Aerobic ◽

Pseudomonas Solanacearum ◽

Triphenyltetrazolium Chloride ◽

Initial Inoculum ◽

Microbial Identification

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-β-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.

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Improved Identification of Mycobacteria by Using the Microbial Identification System in Combination with Additional Trimethylsulfonium Hydroxide Pyrolysis

Journal of Clinical Microbiology ◽

10.1128/jcm.36.9.2477-2480.1998 ◽

1998 ◽

Vol 36 (9) ◽

pp. 2477-2480 ◽

Cited By ~ 40

Author(s):

K.-Dieter Müller ◽

Ernst N. Schmid ◽

Reiner M. Kroppenstedt

Keyword(s):

Sample Preparation ◽

Methyl Esters ◽

Mycolic Acid ◽

Identification System ◽

Mycobacterial Species ◽

Library Search ◽

Microbial Identification ◽

Wide Range ◽

Significant Difference ◽

Microbial Identification System

The MIDI automated Microbial Identification System (MIS) uses gas chromatography (GC) analysis of whole-cell fatty acid methyl esters (FAMEs) between 9 and 20 carbons in length to characterize a wide range of bacterial genera and species, including mycobacteria. Mycolic acid cleavage products (MACPs) with chain lengths of C22 to C26 are not released by MIDI sample preparation of mycobacteria. Therefore, the MIS library search report often matches several mycobacterial species without any significant difference in the similarity indices. The problem is solved by adding trimethylsulfonium hydroxide (TMSH) instead of sodium sulfate in the last step of sample preparation, thus allowing the identification of MACPs in addition to FAMEs. Only one GC run parameter has to be changed: the temperature program must be extended from 260 to 310°C. The MIS library search report for the identification of bacteria is not disturbed by TMSH. The combination of conventional library search report with the information of typical MACP patterns yields significantly better discrimination of mycobacterial species than the MIDI method allows.

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Identification of Three Strains of Mycobacterium Species Isolated from Clinical Samples Using Fatty Acid Methyl Ester Profiling

Journal of International Medical Research ◽

10.1177/147323000303100210 ◽

2003 ◽

Vol 31 (2) ◽

pp. 133-140 ◽

Cited By ~ 13

Author(s):

A Ozbek ◽

O Aktas

Keyword(s):

Fatty Acid ◽

Methyl Esters ◽

Acid Methyl Ester ◽

Cellular Fatty Acid ◽

Clinical Samples ◽

Identification System ◽

Cyclopropane Fatty Acid ◽

Mycobacterium Species ◽

Microbial Identification ◽

Acid Methyl

The cellular fatty acid profiles of 67 strains belonging to three different species of the genus Mycobacterium were determined by gas chromatography of the fatty acid methyl esters, using the MIDI Sherlock® Microbial Identification System (MIS). The species M. tuberculosis, M. xenopi and M. avium complex were clearly distinguishable and could be identified based on the presence and concentrations of 12 fatty acids: 14:0, 15:0, 16:1ω7c, 16:1ω6c, 16:0, 17:0, 18:2ω6,9c, 18:1ω9c, 18:0, 10Me-18:0 tuberculostearic acid, alcohol and cyclopropane. Fatty acid analysis showed that there is great homogeneity within and heterogeneity between Mycobacterium species. Thus the MIS is an accurate, efficient and relatively rapid method for the identification of mycobacteria.

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Occurrence of Bacterial Stem Rot, Caused by Erwinia chrysanthemi, on Field-Grown Tomato in Florida

Plant Disease ◽

10.1094/pdis.1998.82.7.831c ◽

1998 ◽

Vol 82 (7) ◽

pp. 831-831 ◽

Cited By ~ 1

Author(s):

D. O. Chellemi ◽

H. A. Dankers ◽

K. Hill ◽

R. E. Cullen ◽

G. W. Simone ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Methyl Esters ◽

Sole Source ◽

Erwinia Chrysanthemi ◽

Identification System ◽

Bacterial Cells ◽

Sterile Water ◽

Microbial Identification ◽

Pure Cultures ◽

Tomato Isolate

In September 1997, wilted 4-week-old tomato (Lycopersicon esculentum Mill.) plants were observed in a commercial production field in St. Lucie County, FL. Closer inspection of affected plants revealed hollow stems and petioles with dark, water-soaked lesions. Diseased tissue was macerated and streaked onto nutrient agar (NA) and crystal violet pectate (CVP) agar. After incubation for 2 days at 30°C, isolates produced pits on the CVP agar. Isolates were transferred onto NA and the incubation and transfer procedure was performed two additional times to obtain pure cultures. Suspensions of bacterial cells were injected into tomato and tobacco leaves to test for a hypersensitive or pathogenic reaction. Isolates produced collapsed necrotic tissue on tomato while no reaction was observed on tobacco. Tests for differentiating species and subspecies in the ‘carotovora’ group of Erwinia were conducted following the protocol of Dickey and Kelman (1). With known cultures of E. carotovora subsp. carotovora and E. chrysanthemi as controls, the isolate from tomato was determined to function as a facultative anaerobe, utilize asparagine as a sole source of carbon and nitrogen, and give positive reactions for pectate degradation, phosphatase, and growth at 37°C. Known cultures of E. carotovora subsp. carotovora, E. chrysanthemi, and the tomato isolate were grown on trypticase soy broth agar for 24 h at 28°C and their cellular fatty acids derivatized to fatty acid methyl esters (FAMEs). Statistical analyses of FAME profile data (MIDI Microbial Identification System, Newark, DE, version 3.60) identified the tomato isolate as Erwinia chrysanthemi. Pathogenicity was determined by inoculating 50-day-old tomato plants (cv. SunPride) with a suspension of E. chrysanthemi obtained from nutrient broth plates incubated at 24°C for 60 h. Three plants each were inoculated with the E. chrysanthemi identified from tomato, sterile water, and known cultures of E. chrysanthemi and E. carotovora subsp. carotovora by placing a drop at the junction of the petiole and stem and passing a sterile needle through the drop into the stem. Plants were maintained in a greenhouse. Dark, water-soaked cankers were observed on the stems of plants inoculated with E. chrysanthemi, including the tomato isolate and E. carotovora subsp. carotovora, after 7 days. No symptoms were observed on plants inoculated with sterile water. Reisolation of the pathogen and identification was performed with tissue from one of the symptomatic inoculated plants. Analyses of FAMEs confirmed E. chrysanthemi as the causal agent. This is the first report of E. chrysanthemi causing a vascular disease of field-grown tomato in Florida. Reference: (1) R. S. Dickey and A. Kelman. 1988. Pages 44–59 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. N. W. Schaad, ed. American Phytopathological Society, St. Paul, MN.

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Occurrence of Bacterial Stripe of Pearl Millet in Georgia

Plant Disease ◽

10.1094/pdis.2002.86.3.326b ◽

2002 ◽

Vol 86 (3) ◽

pp. 326-326

Author(s):

R. Gitaitis ◽

J. Wilson ◽

R. Walcott ◽

H. Sanders ◽

W. Hanna

Keyword(s):

Pearl Millet ◽

Sweet Corn ◽

Pennisetum Glaucum ◽

Methyl Esters ◽

The United States ◽

Negative Control ◽

Identification System ◽

Breeding Lines ◽

Microbial Identification ◽

Atypical Symptoms

Bacterial stripe, caused by Acidovorax avenae subsp. avenae, was observed on breeding lines of pearl millet (Pennisetum glaucum (L.) R. Br.) in Georgia in 1999 and 2001. A gram-negative, oxidase-positive, rod-shaped bacterium that produced circular, cream-colored, nonfluorescent, butyrous colonies with entire margins on King's medium B was consistently isolated from leaf lesions. The bacterium was identified as A. avenae subsp. avenae by gas-chromatography of extracted, whole-cell, fatty acid methyl esters using the Sherlock Microbial Identification System (MIDI, Newark, DE) and by substrate utilization patterns using the Biolog Identification System (Biolog Inc., Hayward, CA). Isolates from pearl millet produced amplicons of expected size (360 bp) from 16S rDNA after conducting polymerase chain reaction (PCR) with primers WFB1 and WFB2, which are specific for A. avenae. When bacterial suspensions of 1 × 108 CFU/ml were infiltrated into the intercellular spaces of leaves of pearl millet seedlings in the greenhouse, typical water-soaked, reddish-brown stripes developed and were identical to those observed in the field. In contrast to previous reports (1), the pearl millet strains produced atypical symptoms on sweet corn (cvs. Merit and Primetime). Necroses were restricted, lacked customary water-soaking, and were similar to symptoms produced by the watermelon pathogen, A. avenae subsp. citrulli, which was used as a negative control. In contrast, three strains of A. avenae subsp. avenae previously isolated from corn in Georgia produced typical water-soaked stripes in both millet and the sweet corn ‘Merit’. However, like the millet strains, A. avenae subsp. avenae strains from corn produced atypical symptoms on the sweet corn ‘Primetime’. Using immunomagnetic separation and PCR (2), A. avenae subsp. avenae was detected in remaining samples of pearl millet seed planted in Georgia in 2001, as well as in remnant samples of seed sent to Puerto Rico for increase in 2000. The A. avenae subsp. avenae strain recovered from seed was identified by the methods listed above, and in the greenhouse it was identified by the production of typical water-soaked stripes after inoculation of pearl millet. This is the first report of A. avenae subsp. avenae infecting pearl millet in the United States. The detection and distribution of seedborne inoculum in breeding lines is significant since the program at Tifton represents a major effort by the U.S. Department of Agriculture to develop higher-yielding, disease-resistant pearl millet hybrids. Furthermore, the strains from pearl millet appear to be different from previous A. avenae subsp. avenae strains isolated from corn in Georgia, because they did not produce typical disease symptoms when infiltrated in corn leaves. References: (1) L. E. Claflin et al. Plant Dis. 73:1010, 1989. (2) R. R. Walcott and R. D. Gitaitis. Plant Dis. 84:470, 2000.

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Determination of Whole-Cell Fatty Acid Profiles for the Characterization and Differentiation of Isolates of Rhizoctonia solani AG-4 And AG-7

Plant Disease ◽

10.1094/pdis.2000.84.7.785 ◽

2000 ◽

Vol 84 (7) ◽

pp. 785-788 ◽

Cited By ~ 11

Author(s):

R. E. Baird ◽

R. D. Gitaitis ◽

D. E. Carling ◽

S. M. Baird ◽

P. J. Alt ◽

...

Keyword(s):

Fatty Acids ◽

Fatty Acid ◽

Rhizoctonia Solani ◽

Euclidean Distance ◽

Methyl Esters ◽

Principal Component ◽

Identification System ◽

Microbial Identification ◽

Euclidean Distances

Fatty acid methyl esters (FAMEs) of isolates of Rhizoctonia solani AG-4 and AG-7 were characterized by gas chromatography and analyzed with Microbial Identification System software. Palmitic, stearic, and oleic acids were common in all isolates from both anastomosis groups (AGs) and accounted for 95% of the C14 to C18 fatty acids present. Oleic acid, most common in both R. solani AG-4 and AG-7 isolates, accounted for the greatest percentages of total FAMEs. The presence, quantities, or absence of individual fatty acids could not be used for distinguishing AG-4 and AG-7 isolates. Anteisopentadecanoic and 9-heptadecanoic acids, however, were specific to all three AG-7 isolates from Japan but absent in other AG-7 isolates and all AG-4 isolates. Pentadecanoic acid occurred in only two of the R. solani AG-4 isolates, but was not found in any of the AG-7 isolates. The AG-4 isolates could be distinguished from AG-7 isolates when quantities of FAMEs and key FAME ratios were analyzed with cluster analysis and principle components were plotted. Isolates of AG-7 from Arkansas, Indiana, and Georgia appeared to be more closely related to each other than to AG-7 isolates from Japan and Mexico. These differences in FAMEs were sufficiently distinct that isolate geographical variability could be determined. A dendrogram analysis cluster constructed from the FAMEs data showed results similar to that of the principal component analysis. Euclidean distances of total AG-4 isolates were distinct from total AG-7 isolates. The Arkansas and Indiana AG-7 isolates had a similar Euclidean distance to each another but the percentages were different for the AG-7 isolates from Japan and Mexico. In conclusion, variability of the FAMEs identified in this study would not be suitable as the main diagnostic tool for distinguishing individual isolates of R. solaniAG-4 from AG-7.

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