First Report of Crown Gall Caused by an Agrobacterium sp. on Diffuse Knapweed (Centaurea diffusa)

Diffuse knapweed (DK) plants were discovered in Mosier, Wasco County, OR (45.6842°N, 121.4021°W) with crown gall-like symptoms near the soil line. Specimens were collected on 27 July 2004 and sent to the USDA-ARS at Ft. Detrick, MD for identification of disease and pathogen. Pure culture of a bacterium was obtained on potato dextrose agar, and hyperplasia and hypertrophy developed on carrot disks and tomato stems after wound inoculation with a needle contaminated by the agar culture. The same bacterium was reisolated from the galls on DK, thus fulfilling Koch's postulates. Pathogenicity tests involving needle inoculations of stems and petioles resulted in gall formation on Acroptilon repens, Carthamus tinctorius, Centaurea solstitialis, C. maculosa, C. cyanus, Crupina vulgaris, Helianthus annuus, and Rubus armeniacus. In biochemical tests typically used for identification of Agrobacterium species (3), the DK strain grew on D1M agar but not on 2% NaCl medium, produced acid from erythritol but not from melezitose, converted malonic acid to base, and turned litmus milk alkaline. These results are characteristic of Agrobacterium rhizogenes (= Biovar 2), except for the litmus milk reaction. Using 16S rRNA cluster analysis by unweighted pair group method with arithmetic mean (UPGMA, 500 replicates) and basic local alignment search tool (BLAST), the DK strain clustered most closely with A. rubi (GenBank Accession Nos. D12787 and AM181759). The DK strain differed from A. larrymoorei (GenBank Accession No. Z30542), A. tumefaciens (GenBank Accession No. AJ389896), A. rhizogenes (GenBank Accession No. AB247607), and A. vitis (GenBank Accession No. AB247599) on the basis of 16S rRNA sequence cluster analysis. The DK strain differed from A. rubi on the basis of differential reactions with erythritol, litmus milk, and 2% NaCl medium (2,4); and the 16S rRNA sequence of the DK strain differed from that of A. rubi by 11 bp (99.2% similarity). Comparisons also were made between the DK strain and two strains (83A and 135A) of A. tumefaciens (= Biovar 1), described from New Mexico on A. repens (1), a plant species in the same tribe and subtribe of the Asteraceae as DK. Host range reported for the two A. repens strains after artificial greenhouse inoculations was similar to that of the DK strain and it included diffuse knapweed (1). However, 16S sequencing, which confirmed identification of both A. repens strains as A. tumefaciens, showed they differed from the DK strain. The DK strain belongs in the genus Agrobacterium, but it could not be assigned to any known species on the basis of data from phenotypic or 16S sequence comparisons. To our knowledge, this is the first report of crown gall on diffuse knapweed in the field. This strain has been deposited into the International Collection of Phytopathogenic Bacteria at Fort Detrick (Accession No. 60099), and the 16S rRNA sequence has been deposited into the GenBank database (Accession No. EF687663). References: (1) A. J. Caesar, Plant Dis. 78:796, 1994. (2) B. Holmes and P. Roberts, J. Appl. Bacteriol. 50:443, 1981. (3) L. W. Moore et al. Page 17 in: Laboratory Guide for Identification of Plant Pathogenic Bacteria. 3rd ed. N. W. Schaad et al., eds. The American Phytopathological Society, St. Paul, MN, 2001. (4) K. Ophel and A. Kerr, Int. J. Syst. Bacteriol. 40:236, 1990.

Isolamento de Lactobacillusacidophilus a partir de fezes de bezerros

Este experimento foi realizado para isolar, caracterizar e identificar estirpes de Lactobacillusacidophilus, a partir de fezes de bezerros com 1 - 3 dias de idade, coletadas diretamente no reto. Os seguintes testes para caracterização dos isolados foram feitos: morfologia celular, teste de gram, catalase, produção de gás a partir da glicose, crescimento a 15 e 45ºC, redução do "Litmus milk", crescimento no meio com 4 e 8% de cloreto de sódio, hidrólise de arginina, redução do nitrato e fermentação de diferentes "carboidratos". De um total de 526 "isolados" iniciais, 12 (2,28%) estirpes de Lactobacillusacidophlilus foram identificadas.

Comparison of Simple and Rapid Methods for Identifying Enterococci Intrinsically Resistant to Vancomycin

Three different methodologies, reduction of litmus milk (LM) and acidification of arabinose (ARA), acidification of methyl-α-d-glucopyranoside (MGP), and rapid motility (RM), for differentiating isolates of Enterococcus casseliflavus and Enterococcus gallinarum(intrinsically vancomycin-resistant enterococci [IVRE]) fromEnterococcus faecalis and Enterococcus faeciumwere evaluated. All 33 isolates of E. faecalis tested reduced LM within 4 h and were negative in all other tests, while the 53 isolates of E. faecium were ARA positive only. In contrast, 45 of 46 (98%) IVRE isolates examined (26 E. casseliflavus and 20 E. gallinarum isolates) acidified MGP, 41 of 46 (89%) were LM and ARA positive, and 45 of 46 (98%) were RM positive. Acidification of MGP was therefore the single most useful test for differentiating IVRE from vancomycin-resistantE. faecium and E. faecalis; however, a combination of LM-ARA and RM testing enabled the correct designation of organisms without the need for overnight incubation.

Potato-Like Odor of Retail Beef Cuts Associated with Species of Pseudomonas

Retail cuts of beef and hamburger packages from a North Dakota meat processor were examined due to consumer complaints of a strong potato-like or musty odor associated with the meat. Examination for total numbers of aerobic bacteria on plate count agar and for gram-negative psychrotrophic bacteria on crystal violet tetrazolium agar revealed numbers in excess of 108 CFU/g. Numbers of coliform bacteria on violet red bile agar were in excess of 106 CFU/g. Gram-negative rods were isolated and identified. The isolates were characterized by a positive catalase reaction, oxidase production, an oxidative O/F reaction, nonutilization of lactose, liquefication of nutrient gelatin, slight motility, production of acid in litmus milk with decoloration and clotting, nonproduction of indole, and nonreduction of nitrate. The isolate was tentatively identified as a Pseudomonas of undetermined species, probably a variant of either Pseudomonas taetrolens or Pseudomonas perolens.

Direct Testing of Gelatin Hydrolysis in Rapid Perfringens Medium

Rapid perfringens medium (RPM) was previously shown to be effective for detection of low numbers of Clostridium perfringens in foods. The detection system of RPM is based on a stormy fermentation of litmus milk. RPM also contains 6% gelatin, and the present work was performed to determine if gelatin hydrolysis can be tested directly in this medium. Twenty-three strains of C. perfringens and related species were inoculated into tubes of RPM and lactose gelatin. All strains were positive for stormy fermentation on RPM after 24 h incubation at 46°C. Seventeen exhibited gelatin hydrolysis on both RPM and lactose gelatin. Tubes of RPM inoculated with slurries of food samples spiked with each of 11 strains of C perfringens were positive for stormy fermentation and gelatin liquefaction. Most C. perfringens hydrolyze gelatin, so this test augments stormy fermentation in RPM as an additional indicator for the presence of this pathogen.

Survival and Growth of Clostridium Species in the Presence of Hydrogen Peroxide

Some bacteria in the genus Clostridium can occur as contaminants in milk. If cheese is made from milk with such contaminants, the bacteria can cause the “late gas” or “late blowing” defect in the cheese. Since hydrogen peroxide can be used to treat milk for cheesemaking, this investigation was initiated to determine effects of the peroxide on viability and growth of Clostridium tyrobutyricum NIZO, C. tyrobutyricum 144, Clostridium perfringens 115 and Clostridium sporogenes T9. Presence of 0.01% hydrogen peroxide in litmus milk retarded but did not prevent growth of and gas production by the clostridia. Presence of 0.02% peroxide inhibited growth and gas production when litmus milk contained, per milliliter, 50 or 100 spores of any of the clostridia being studied. These numbers of clostridial spores are greater than those normally found in raw milk produced under ordinary conditions.

Occurrence of Bacillus cereus and the Bacteriological Quality of Chinese “Take-Out” Foods

One hundred sixty-five samples of various foods were collected from 24 different Chinese take-out restaurants for bacteriological examination which included enumeration of Bacillus cereus by three media, MYP, KG and blood agars. Blood agar was less selective but no quantitative differences in recovery were apparent. Twenty-eight samples (15%) yielded B. cereus in excess of 100 per gram, and 20 of these were fried rice (33% positive), which also showed the poorest overall bacteriological quality. Biochemical characterization of 232 isolates of B. cereus showed 96% or more positive for catalase, nitrate reduction, beta-haemolysis, subterminal-ellipsoidal spores, aerobic and anaerobic utilization of glucose, Voges-Proskauer, fermentation of glycerol, gelatin hydrolysis, and alkaline peptonization of litmus milk; and a negative reaction in mannitol. Variable results were obtained for motility, fermentation of sucrose and salicin, and starch hydrolysis. Thirty-three isolates were susceptible to 12 of 19 antibiotics tested, and resistant to colistin. Six (18%) were susceptible to penicillin.

Use of Litmus Milk Agar for Presumptive Identification of Cutaneous Propionibacteria

Presumptive identification of cutaneous propionibacteria can be achieved with litmus milk agar as medium.