Identification (based on Membrane Fatty Acid Methyl Ester Analysis and Partial Sequencing of 16s Ribosomal Rna) of Bacterial Strains Used in the Biological Control of Fusarium Head Blight

Our laboratory has been working for the last several years with bacterial strains (designated as 1B-A, 1B-C, 1B-E, and 1D-3) isolated from South Dakota wheat foliage and residue which are able to antagonize Fusarium graminearum in laboratory plate assays and in field plot trials. Although we have known for many years that the bacterial strains are endospore formers that are able to grow aerobically, likely being members of the genus Bacillus, the exact identity of the strains has remained problematic. Systematics of the genus Bacillus have undergone great changes since modern methods for bacterial identification, such as analysis of membrane fatty acid methyl esters (FAME analysis) and analysis of small subunit 16S ribosomal RNA sequences have become available. Analysis of FAME patterns of the four strains done about six years ago suggested that bacterial strain 1D-3 was almost certainly Bacillus amyloliquefaciens, whereas the other three strains were not extremely similar to any bacteria in the FAME database, but were related to a degree to Bacillus atrophaeus (formerly B. subtilis variety niger). This year another laboratory did FAME analyses on the four strains. Results strongly indicated that strains 1B-A and 1D-3 were Bacillus lentimorbus, and that strains 1B-E and 1B-C were Bacillus subtilis. In addition, the first 500 base pairs of the 16S rRNA gene of each strain were sequenced and compared to known sequences of ribosomal RNA genes, and alignment profiles and phylogenetic trees were derived from the sequences. Usually knowing the first 500 base pairs of the 16S rRNA gene is sufficient to determine the identity of most bacterial strains. All four strains (1B-A, 1B-C, 1B-E, and 1D-3) had identical sequences in the first 500 base pairs of their 16S RNA genes, and all were most closely related to Bacillus amyloliquefaciens and had less but significant relatedness to Bacillus atrophaeus. This and other studies have found that FAME analysis will not necessarily agree with the results of 16S rRNA sequencing. Extent and completeness of the database used in each taxonomic analysis is extremely important when attempting to identify a bacterial strain, and in some cases (such as this study) further standard physiologic tests will be needed to help make a confident identification of the bacterial strains. Complete 16S RNA sequences of the strains would be very valuable in helping to determine whether the strains truly belong to known bacterial species, or are one or more new species in the genus Bacillus. In addition, detailed physiologic tests will be conducted to help further evaluate the relatedness of these bacterial strains to known bacterial species. Thorough understanding of the enzymatic activities of these strains will help optimize their formulation and application as biological control agents used to control FHB in the field. 2001 National Fusarium Head Blight Forum 46 Chemical and Biological Control BIODIVERSITY OF MICROBIAL ANTAGONISTS TO GIBBERELLA ZEAE IN BRAZIL Wilmar C. da Luz Embrapa Trigo, Caixa Postal 451, 900-970, Passo Fundo, RS, Brazil Corresponding Author: PH: (55) 54 311 3444; E-mail: [email protected] OBJECTIVES To determine the diversity of microbial antagonistic agents to Gibberella zeae. INTRODUCTION Knowledge of biodiversity of living organisms is mainly important to determine their potential functions. Fusarium head blight (FHB), induced by Gibberella zeae (anamorph = Fusarium graminearum Schw.) is a prevalent wheat disease in Brazil, causing crop production losses varying from 10% (Luz, 1984); to 54 % (Picinini & Fernandes, 1994). Due to the difficulty in controlling the disease by chemical treatment, crop rotation and varietal resistance, biological control agents are beeing evaluated as an additional tactics for integrated management of FHB. To have an idea of potential bioprotectants, biodiversity of promining isolates need to be established. MATERIALS AND METHODS Thousands of microorganisms were screened in vitro and in vivo against G. zeae. Biodiversity of most effective microbial strains was established by systematic determination using physiological, biochemical and morphological features as well as systems such as Biolog, GC-Fame (analysis of fat acids) and comparison of the sequences of the small subunits of the RNA 16S (for bacterial strains), comparing 500 base pairs. RESULTS AND DISCUSSION The diversity of microorganisms that shows potential for managing FHB in Brazil comprises 15 different species as presented in Table 1. Searching different isolates of these microorganisms with different degree of control efficiency may be of valuable interest to investigators in other countries as well as in Brazil. This microbiota may have an important impact in inproving FHB control either alone or as an additional measure to supplement chemical, cultural and resistance control methods. 2001 National Fusarium Head Blight Forum 47 Chemical and Biological Control Table 1. Biodiversity of microorganisms for biocontrol of Fusarium head blight Wheat in Brazil 1 Identifications of bacterial isolates by Microbe Inotech Laboratories, Inc. Saint Louis, Mo, USA, based on GCFAME, Biolog, and/or 16Sr RNA sequence. Bacterial colony morphology, physiological tests, and morphological indentification of fungi by Plant Pathology Laboratory, Embrapa Trigo, Passo Fundo, RS, Brazil.