No abstract

Aims: To taxonomically position two bacterial strains conferring biological control activity towards plant diseases. Methods and Results: Key phenotypic characteristics, including gliding motility and a high percentage of G + C content, indicated biocontrol strains N4-7 and C3 were essentially identical to those described for Lysobacter enzymogenes. Cellular fatty acid analysis confirmed a close relatedness of strains N4-7 and C3 to L. enzymogenes and a more distant relatedness to L. antibioticus. The 16S rDNA phylogenetic analysis revealed a distinct Lysobacter clade that included both strains within the c-proteobacteria. Conclusions: The combined taxonomic methods provide clear evidence that N4-7 and C3 should be grouped as strains of L. enzymogenes and not Stenotrophomonas maltophilia or a novel taxon. Phylogenetic analysis of 16S rDNA formed a Lysobacter clade that included several other environmentally diverse bacterial strains obtained from databases and confirmed relatedness of strains N4-7 and C3 to L. enzymogenes. Significance and Impact of the Study: Inclusion of N4-7 and C3 as strains of L. enzymogenes is among the first description of members of this genus as biocontrol agents of plant diseases. These results suggest that members of the Lysobacter group might provide a new source as plant-associated microbes that display biocontrol activity.

Lysobacter enzymogenes strain N4-7 produces multiple biochemically distinct extracellular ␤-1,3-glucanase activities. The gluA, gluB, and gluC genes, encoding enzymes with ␤-1,3-glucanase activity, were identified by a reverse-genetics approach following internal amino acid sequence determination of ␤-1,3-glucanase-active proteins partially purified from culture filtrates of strain N4-7. Analysis of gluA and gluC gene products indicates that they are members of family 16 glycoside hydrolases that have significant sequence identity to each other throughout the catalytic domain but that differ structurally by the presence of a family 6 carbohydrate-binding domain within the gluC product. Analysis of the gluB gene product indicates that it is a member of family 64 glycoside hydrolases. Expression of each gene in Escherichia coli resulted in the production of proteins with ␤-1,3-glucanase activity. Biochemical analyses of the recombinant enzymes indicate that GluA and GluC exhibit maximal activity at pH 4.5 and 45°C and that GluB is most active between pH 4.5 and 5.0 at 41°C. Activity of recombinant proteins against various ␤-1,3 glucan substrates indicates that GluA and GluC are most active against linear ␤-1,3 glucans, while GluB is most active against the insoluble ␤-1,3 glucan substrate zymosan A. These data suggest that the contribution of ␤-1,3-glucanases to the biocontrol activity of L. enzymogenes may be due to complementary activities of these enzymes in the hydrolysis of ␤-1,3 glucans from fungal cell walls.Members of the genus Lysobacter typically are found in soil and water habitats and are characterized by gliding motility and the ability to lyse other microorganisms, including fungi and nematodes (4). One species within this group, Lysobacter enzymogenes, produces an impressive repertoire of extracellular degradative enzyme activities. These include chitinase, glucanase, and protease activities, which can degrade components found in fungal cell walls and which are presumed to contribute to the lytic activity of L. enzymogenes. Despite these described traits, relatively little is known about the hydrolytic enzymes that L. enzymogenes produces or the contributing role for each enzyme class in the lytic activity of L. enzymogenes. To date, only proteases (7, 33, 40) from the species have been characterized at the molecular and biochemical levels.␤-1,3-Glucanases, which hydrolyze glucan polymers containing ␤-1,3 linkages (3, 39) from a number of microbial species, have been characterized. Proposed physiological functions for these enzymes vary depending on the source of enzyme. Among bacteria, many ␤-1,3-glucanases have been studied for purposes of fungal and yeast cell wall degradation (see, e.g., references 2, 9, 17, 27, 28, 31, and 41). However, despite obvious associations with fungal antagonism, few ␤-1,3-glucanases from bacteria with demonstrated biological control activity toward fungal plant pathogens have been characterized. The bacterial biocontrol strain N4-7 was isolated originally as a biocont...

Ampelomyces and Phoma species are frequently confused with each other. Isolates previously attributed to the genus Ampelomyces were shown to be Phoma isolates through studies of their morphology and life cycle and ribosomal DNA internal transcribed spacer region 1 sequence analysis. Phoma glomerata can colonize and suppress development of powdery mildew on oak and may have utility as a mycoparasitic agent.Powdery mildews are widespread plant pathogens that are conspicuous by their white mycelia and powder-like conidia (20). The fungus Ampelomyces quisqualis Ces. is the only fungus that has been demonstrated to be generally effective as a biocontrol agent of powdery mildew (4, 9, 16). Many morphologically similar species may be confused with A. quisqualis (10). To evaluate this possibility, we examined and identified Ampelomyces-like fungi isolated from powdery mildew and compared these cultures with isolates identified as Ampelomyces in culture collections.Isolation and growth. Leaves of sycamore trees (Platanus occidentalis L.) bearing infections of powdery mildew (Microsphaera penicillata (Wallr.:Fr.) Lèv.) were located in South River, New Jersey, in July 1998. Microscopic examination of the leaves revealed two types of pycnidia: stipitate pycnidia, typical of A. quisqualis, and sessile pycnidia, typical of the genus Phoma (Fig. 1) (17). Both types of pycnidia were removed from leaves with fine needles and placed on potato dextrose agar (Difco, Inc., Detroit, Mich.) containing the antibiotics gentamicin (40 mg/liter), streptomycin (40 mg/liter), and penicillin (20 mg/liter) (PDA ϩ 3). Two different fungi were consistently recovered. The stipitate pycnidia developed into slow-growing colonies whose characteristics corresponded to those expected for A. quisqualis (5, 11). The sessile pycnidia developed into rapidly growing colonies whose characteristics corresponded to those of Phoma glomerata (Cda) Wollenw. (2,19).Agar plugs (6 mm in diameter) of mycelia cut from the margins of rapidly growing colonies of both the South River Ampelomyces and South River P. glomerata isolates were transferred to five plates each of PDAϩ3 and incubated at room temperature (21 to 22°C) for 3 weeks to measure growth rates. We measured an average growth of 8 Ϯ 1 mm/day for the P. glomerata isolates and an average growth of 0.8 Ϯ 0.1 mm/day for the Ampelomyces isolates. With age, cultures of P.

We report the occurrence of Bacillus amyloliquefaciens in vanilla orchids (Vanilla phaeantha) and cultivated hybrid vanilla (V. planifolia × V. pompona) as a systemic bacterial endophyte. We determined with light microscopy and isolations that tissues of V. phaeantha and the cultivated hybrid were infected by a bacterial endophyte and that shoot meristems and stomatal areas of stems and leaves were densely colonized. We identified the endophyte as B. amyloliquefaciens using DNA sequence data. Since additional endophyte-free plants and seed of this orchid were not available, additional studies were performed on surrogate hosts Amaranthus caudatus, Ipomoea tricolor, and I. purpurea. Plants of A. caudatus inoculated with B. amyloliquefaciens demonstrated intracellular colonization of guard cells and other epidermal cells, confirming the pattern observed in the orchids. Isolations and histological studies suggest that the bacterium may penetrate deeply into developing plant tissues in shoot meristems, forming endospores in maturing tissues. B. amyloliquefaciens produced fungal inhibitors in culture. In controlled experiments using morning glory seedlings we showed that the bacterium promoted seedling growth and reduced seedling necrosis due to pathogens. We detected the gene for phosphopantetheinyl transferase (sfp), an enzyme in the pathway for production of antifungal lipopeptides, and purified the lipopeptide "surfactin" from cultures of the bacterium. We hypothesize that B. amyloliquefaciens is a robust endophyte and defensive mutualist of vanilla orchids. Whether the symbiosis between this bacterium and its hosts can be managed to protect vanilla crops from diseases is a question that should be evaluated in future research.