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Pseudomonas fluorescens strains that produce the polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are among the most effective rhizobacteria that suppress root and crown rots, wilts, and damping-off diseases of a variety of crops, and they play a key role in the natural suppressiveness of some soils to certain soilborne pathogens. Root colonization by 2,4-DAPG-producing P. fluorescens strains Pf-5 (genotype A), Q2-87 (genotype B), Q8r1-96 (genotype D), and HT5-1 (genotype N) produced induced systemic resistance (ISR) in Arabidopsis thaliana accession Col-0 against bacterial speck caused by P. syringae pv. tomato. The ISR-eliciting activity of the four bacterial genotypes was similar, and all genotypes were equivalent in activity to the well-characterized strain P. fluorescens WCS417r. The 2,4-DAPG biosynthetic locus consists of the genes phlHGF and phlACBDE. phlD or phlBC mutants of Q2-87 (2,4-DAPG minus) were significantly reduced in ISR activity, and genetic complementation of the mutants restored ISR activity back to wild-type levels. A phlF regulatory mutant (overproducer of 2,4-DAPG) had ISR activity equivalent to the wild-type Q2-87. Introduction of DAPG into soil at concentrations of 10 to 250 μM 4 days before challenge inoculation induced resistance equivalent to or better than the bacteria. Strain Q2-87 induced resistance on transgenic NahG plants but not on npr1-1, jar1, and etr1 Arabidopsis mutants. These results indicate that the antibiotic 2,4-DAPG is a major determinant of ISR in 2,4-DAPG-producing P. fluorescens, that the genotype of the strain does not affect its ISR activity, and that the activity induced by these bacteria operates through the ethylene- and jasmonic acid-dependent signal transduction pathway.

Kwak, Y.-S., Bakker, P. A. H. M., Glandorf, D. C. M., Rice, J. T., Paulitz, T. C., and Weller, D. M. 2010. Isolation, characterization, and sensitivity to 2,4-diacetylphloroglucinol of isolates of Phialophora spp. from Washington wheat fields. Phytopathology 100:404-414. Dark pigmented fungi of the Gaeumannomyces–Phialophora complex were isolated from the roots of wheat grown in fields in eastern Washington State. These fungi were identified as Phialophora spp. on the basis of morphological and genetic characteristics. The isolates produced lobed hyphopodia on wheat coleoptiles, phialides, and hyaline phialospores. Sequence comparison of internal transcribed spacer regions indicated that the Phialophora isolates were clearly separated from other Gaeumannomyces spp. Primers AV1 and AV3 amplified 1.3-kb portions of an avenacinase-like gene in the Phialophora isolates. Phylogenetic trees of the avenacinase-like gene in the Phialophora spp. also clearly separated them from other Gaeumannomyces spp. The Phialophora isolates were moderately virulent on wheat and barley and produced confined black lesions on the roots of wild oat and two oat cultivars. Among isolates tested for their sensitivity to 2,4-diacetylphloroglucinol (2,4-DAPG), the 90% effective dose values were 11.9 to 48.2 µg ml–1. A representative Phialophora isolate reduced the severity of take-all on wheat caused by two different isolates of Gaeumannomyces graminis var. tritici. To our knowledge, this study provides the first report of an avenacinase-like gene in Phialophora spp. and demonstrated that the fungus is significantly less sensitive to 2,4-DAPG than G. graminis var. tritici.

Ran, L. X., van Loon, L. C., and Bakker, P. A. H. M. 2005. No role for bacterially produced salicylic acid in rhizobacterial induction of systemic resistance in Arabidopsis. Phytopathology 95:1349-1355. The role of bacterially produced salicylic acid (SA) in the induction of systemic resistance in plants by rhizobacteria is far from clear. The strong SA producer Pseudomonas fluorescens WCS374r induces resistance in radish but not in Arabidopsis thaliana, whereas application of SA leads to induction of resistance in both plant species. In this study, we compared P. fluorescens WCS374r with three other SA-producing fluorescent Pseudomonas strains, P. fluorescens WCS417r and CHA0r, and P. aeruginosa 7NSK2 for their abilities to produce SA under different growth conditions and to induce systemic resistance in A. thaliana against bacterial speck, caused by P. syringae pv. tomato. All strains produced SA in vitro, varying from 5 fg cell –1 for WCS417r to >25 fg cell –1 for WCS374r. Addition of 200 µM FeCl3 to standard succinate medium abolished SA production in all strains. Whereas the incubation temperature did not affect SA production by WCS417r and 7NSK2, strains WCS374r and CHA0r produced more SA when grown at 33 instead of 28°C. WCS417r, CHA0r, and 7NSK2 induced systemic resistance apparently associated with their ability to produce SA, but WCS374r did not. Conversely, a mutant of 7NSK2 unable to produce SA still triggered induced systemic resistance (ISR). The possible involvement of SA in the induction of resistance was evaluated using SA-nonaccumulating transgenic NahG plants. Strains WCS417r, CHA0r, and 7NSK2 induced resistance in NahG Arabidopsis. Also, WCS374r, when grown at 33 or 36°C, triggered ISR in these plants, but not in ethylene-insensitive ein2 or in non-plant pathogenesis-related protein-expressing npr1 mutant plants, irrespective of the growth temperature of the bacteria. These results demonstrate that, whereas WCS374r can be manipulated to trigger ISR in Arabidopsis, SA is not the primary determinant for the induction of systemic resistance against bacterial speck disease by this bacterium. Also, for the other SAproducing strains used in this study, bacterial determinants other than SA must be responsible for inducing resistance.

Transgenic tobacco plants (Tetr) expressing the mutant etr1-1 gene from Arabidopsis thaliana are insensitive to ethylene and develop symptoms of wilting and stem rot when grown in nonautoclaved soil. Several isolates of Fusarium, Thielaviopsis, and Pythium were recovered from stems of diseased Tetr plants. Inoculation with each of these isolates of 6-week-old plants growing in autoclaved soil caused disease in Tetr plants but not in nontransformed plants. Also, when 2-week-old seedlings were used, nontransformed tobacco appeared nonsusceptible to the Fusarium isolates, whereas Tetr seedlings did develop disease. Tetr seedlings were not susceptible to several nonhost Fusarium isolates. In contrast to results with Fusarium isolates, inoculation of 2-week-old seedlings with a Thielaviopsis isolate resulted in equal symptom development of nontransformed and Tetr tobacco. In order to explore the potential range of pathogens to which Tetr tobacco plants display enhanced susceptibility, the pathogenicity of several root and leaf pathogens was tested. Tetr plants were more susceptible to the necrotrophic fungi Botrytis cinerea and Cercospora nicotianae and the bacterium Erwinia carotovora, but only marginally more to the bacterium Ralstonia solanacearum. In contrast, the biotrophic fungus Oidium neolycopersici, the oomycete Peronospora tabacina, and Tobacco mosaic virus caused similar or less severe symptoms on Tetr plants than on nontransformed plants. Total peroxidase activity of Tetr plants was lower than that of nontransformed plants, suggesting a role for peroxidases in resistance against necrotrophic microorganisms. A comparable range of pathogens was examined on Arabidopsis and its ethylene-insensitive mutants etr1-1 and ein2-1. With the exception of one Fusarium isolate, ethylene insensitivity increased susceptibility of Arabidopsis plants to a similar spectrum of necrotizing pathogens as in tobacco. Thus, both ethylene-insensitive tobacco and Arabidopsis plants appear to be impaired in their resistance to necrotrophic pathogens.

During the last few years, Kloepper and coworkers have presented impressive evidence that plant growth-promoting rhizobacteria (PGPR) can protect cucumber plants against fungal, bacterial, and viral diseases. After Wei et al. (14) described induction of systemic resistance of cucumber to Colletotrichum orbiculare by six strains of PGPR, it was repeatedly stated in abstracts of meetings and book chapters that PGPR strains with previously demonstrated induced systemic resistance (ISR) against C. orbiculare led to multiple-pathogen control. However, these strains were not specified, and the communications lacked the technical details necessary to judge whether ISR was the (sole) mechanism responsible. Only recently, two PGPR strains were described as inducing resistance against Fusarium wilt (7), angular leaf spot (8), and anthracnose (9), but the strains had different designations and were not among those used previously (14). Hence, we at first concluded that proof still needed to be provided that the same inducing bacterium could suppress diseases caused by different infectious agents through ISR. Our experiments were conducted before Liu et al. (9) provided details about the strains inducing ISR against C. orbiculare. Nevertheless, we now see that the same PGPR strains did indeed protect against a foliar fungus, a soilborne fungus, and a bacterium, and we admit that we overlooked these recent results when submitting our paper. To conclude that the mechanism responsible for suppression is ISR requires that the disease suppression is shown to be plant mediated and that it extends to plant parts that are not in contact with the inducing agent. This was neatly demonstrated for one PGPR strain in plants protected against Fusarium wilt, because a bioluminescent derivative of strain 89B-27 applied to one part of a split root system did not move to the part inoculated with Fusarium oxysporum f. sp. cucumerinum (7). The other studies did not address this question in detail, although it was mentioned that bacteria inducing systemic resistance were not translocated and did not colonize challenged leaves (6,8). We agree that this implies ISR involvement. However, application of PGPR by seed treatment or cotyledon injection does not exclude colonization of the foliar parts of plants by the bacteria, and hence, microbial antagonism also might play a role. In our studies on ISR in carnation (11), radish (3), and Arabidopsis (10), we have repeatedly shown that inducing bacteria were not recoverable from sites where plants were challenged and that the spatial separation was maintained for the duration of the experiments. Moreover, in all three plant species, ISR was induced by preparations of the bacterial lipopolysaccharide (4,12,13), ruling out any protective effects due to bacterial metabolism. Both Kloepper and coworkers and we have shown that similar strains of PGPR protect various plant species against diseases caused by different types of pathogens, and in doing so, both groups have independently validated the other’s results. PGPR-mediated ISR is an important mechanism of biological disease control, and it can be as effective as pathogen-induced systemic acquired resistance (1,2). Both Leeman et al. (5) and Wei et al. (15) have shown that PGPR are effective in reducing diseases under field conditions. This makes it even more important to further characterize the physiological, biochemical, and molecular mechanisms involved to make optimal use of ISR in plant protection.