Evaluation of peracid formation as the basis for resistance to infection in plants transformed with haloperoxidase.

Nonheme haloperoxidase (HPO-P) isolated from Pseudomonas pyrrocinia catalyzed the peroxidation of alkyl acids to peracids. Among acids tested as substrates, acetic acid was most readily peroxidized. The reaction product peracetate possessed potent antifungal activity: 50% death (LD(50)) of Aspergillus flavus occurred at 25 microM peracetate. Viability of A. flavus was inhibited by up to 80% by leaf extracts of tobacco plants transformed with the HPO-P gene from P. pyrrocinia compared to viability of fungi exposed to extracts from controls. To elucidate if peracid formation by HPO-P was the basis for antifungal activity in transgenic leaf tissues, lethalities of hydrogen peroxide-acetate-HPO-P combinations against A. flavus were examined in vitro. LD(50) of A. flavus exposed to the combinations occurred at 30 mM acetate when concentrations of hydrogen peroxide and HPO-P were held constant. This value was identical to the LD(50) produced by 30 mM acetate in the absence of hydrogen peroxide-HPO-P and therefore did not account for enhanced antifungal activity in transgenic plants. For clarification, kinetics of the enzymic reaction were examined. According to the concentration of acetate needed for enzyme saturation (K(m) = 250 mM), acetate was lethal prior to its oxidation to peracetate. Results indicate that peracid generation by HPO-P was not the basis for enhanced antifungal activity in transgenic plants expressing the HPO-P gene.

Broad-spectrum antimicrobial activity in vitro of the synthetic peptide D4E1.

Broad-spectrum antimicrobial activity of a synthetic peptide, D4E1, is documented in this paper. D4E1 inhibited the growth of several fungal phytopathogens belonging to four classes-Ascomycetes, Basidiomycetes, Deuteromycetes, and Oomycetes, and two bacterial pathogens, Pseudomonas syringae pv. tabaci and Xanthomonas campestris pv. malvacearum race 18. The minimum inhibitory concentration (MIC) of D4E1 required to completely inhibit the growth of all fungi studied ranged from 4.67 to 25 microM. Fungal pathogens highly sensitive to D4E1 include Thielaviopsis basicola, Verticillium dahliae, Fusarium moniliforme, Phytophthora cinnamomi, and Phytophthora parasitica. Comparatively, the least sensitive fungal pathogens were Alternaria alternata, Colletotrichum destructivum, and Rhizoctonia solani. The two bacterial pathogens, P. syringae pv. tabaci and X. campestris pv. malvacearum race 18, were most sensitive to D4E1 with MIC values of 2.25 and 1.25 microM, respectively. Microscopic analysis of D4E1 effects on fungal morphology of Aspergillus flavus and R. solani revealed abnormal hyphal growth and discontinuous cytoplasm. After 8 h of exposure to 25 microM D4E1, A. flavus spore germination was reduced by 75%. The suitability of peptide D4E1 to enhance disease resistance in transgenic crop plants is discussed.

Inhibition of fungal growth in planta and in vitro by transgenic tobacco expressing a bacterial nonheme chloroperoxidase gene.

Transgenic tobacco plants producing chloroperoxidase (CPO-P), encoded by a novel gene from Pseudomonas pyrrocinia, were obtained by Agrobacterium-mediated transformation. Successful transformation was shown by PCR, Southern, northern and western blot analyses, and assays of CPO-P enzyme activity. Extracts from plants transformed with the CPO-P gene significantly reduced Aspergillus flavus colonies by up to 100% compared with extracts from control plants transformed with pBI121. Compared with controls, the transformed plants showed increased disease resistance in planta against a fungal pathogen, Colletotrichum destructivum, the causal agent of tobacco anthracnose.

Relationship Between Phyllosphere Population Sizes of Xanthomonas translucens pv. translucens and Bacterial Leaf Streak Severity on Wheat Seedlings.

ABSTRACT The relationship between leaf-associated population sizes of Xanthomonas translucens pv. translucens on asymptomatic leaves and subsequent bacterial leaf streak (BLS) severity was investigated. In three experiments, X. translucens pv. translucens was spray-inoculated onto 10-day-old wheat seedlings over a range of inoculum densities (10(4), 10(5), 10(6), 10(7), and 10(8) CFU/ml). Lesions developed most rapidly on plants inoculated with higher densities of X. translucens pv. translucens. Leaf-associated pathogen population sizes recovered 48 h after inoculation were highly predictive of BLS severity 7 days after inoculation (R(2) = 0.970, P < 0.0001). The relationship between pathogen population size on leaves and subsequent BLS severity was best described by the logistic model. Leaf-associated X. translucens pv. translucens population size and BLS severity from a particular pathogen inoculum density often varied among experiments; however, the disease severity level caused by a particular leaf-associated X. translucens pv. translucens population size was not significantly different among experiments. Biological and disease control implications of the X. translucens pv. translucens population size-BLS severity relationship are discussed.