Exploration of the Use of the “Bialaphos Genes” for Improving Bioherbicide Efficacy

Abstract

We are studying the possibility of altering the virulence and host range of a phytopathogen by transferring and expressing certain genes from the soil-dwelling saprophyte, Streptomyces hygroscopicus, in a plant pathogen model, Xanthomonas campestris pv. campestris (XCC). The genes, referred to herein as the “bialaphos genes,” encode the production of bialaphos, a potent glutamine-synthetase-inhibiting herbicide. This cluster of genes was originally isolated from several biosynthetically blocked mutants of S. hygroscopicus and constructed into a plasmid vector, pBG9. We have transferred a fragment of the gene cluster into pLAFR3, a plasmid that functions in both Escherichia coli and XCC and contains a tetracycline resistance marker. The resulting plasmid, named pIL-1, was used to transform E. coli and was incorporated into XCC by conjugation. The transfer of the fragment was confirmed by Southern analysis. The genes were maintained in XCC for about 47 generations in the absence of selection for tetracycline, and no changes in cultural phenotypes were seen in the transformed XCC (XCC/pIL-1). The XCC/pIL-1 cells were pathogenic to their natural hosts cabbage and broccoli, but induced an altered hypersensitive response in the nonhosts bean, pepper, sunflower, and tobacco. The pathogenic host-reaction, induced by the parent XCC, XCC/pLAFR3, and XCC/pIL-1, was a typical black rot disease in inoculated leaves of the two hosts. The nonhost reaction on the nonhost leaves was necrotic hypersensitivity, induced by XCC and XCC/pLAFR3, or the inhibition of hypersensitivity accompanied by only chlorosis at sites inoculated with XCC/pIL-1. We hypothesize that the altered hypersensitivity phenotype may be due to the transformed XCC becoming more compatible with the nonhosts, a step toward acquiring nonhost-virulence, or due to disruption of the normal expression of the hypersensitivity and pathogenicity genes in the transformed XCC. More work is needed to confirm that the introduced genes are being expressed in XCC. With further understanding, this approach may provide a useful model to study host range, virulence, and strain improvement of plant pathogens for biological control of weeds.

Where are the bioherbicides?

“…However, most studies and discussions have focused on the enhancement of virulence (Amsellem et al 2002;Gressel 2002;Vurro et al 2001), which has primarily focused on the enhancement of the production of an enzyme or toxin (Greaves et al 1989;Gressel 2002). In addition, a pathogenic or nonpathogenic microbe could be used to colonize a weed and deliver a herbicidal molecule (Charudattan et al 1996). Fungi such as Fusarium spp.…”

“…Only a few examples of microbes genetically engineered for enhanced bioherbicide potential have been reported. Charudattan et al (1996) investigated the use of generating enhanced Xanthomonas isolates by transformation with genes encoding bialaphos and found that transgenic isolates were more virulent than the wild type. A number of groups have studied NEP1, a gene encoding ''necrosis eliciting protein'' (Amsellem et al 2002;Bailey et al 1997Bailey et al , 2000, and this model has proven valuable.…”

Microbial agents for control of aquatic weeds and their role in integrated management.

“…For example, Tiourebev et al [111] have attempted a new approach to enhance virulence of a microbial agent by selecting strains that are capable of producing high levels of amino acids that can suppress the growth and development of plants causing leaf distortion, loss of apical dominance and stunted growth. Charudattan et al [112] altered the virulence and host range of a bacterium by inserting genes, which encoded for the production of bialaphos, a glutamine-synthetase inhibiting herbicide. Alteration of such bacterial genes could produce overall increases in virulence, host range or other related traits.…”

Microbial Allelochemicals and Pathogens as Bioherbicidal Agents1

“…Work on the use of bialaphos genes is being performed Volume 15, Issue 4 (October-December) 2001 by transforming Xanthomonas campestris pv. campestris with bialaphos genes (Charudattan et al 1996). Alteration of such bacterial genes could produce overall increases in virulence, host range, or other related traits.…”