Particular bacterial strains in certain natural environments prevent infectious diseases of plant roots. How these bacteria achieve this protection from pathogenic fungi has been analysed in detail in biocontrol strains of fluorescent pseudomonads. During root colonization, these bacteria produce antifungal antibiotics, elicit induced systemic resistance in the host plant or interfere specifically with fungal pathogenicity factors. Before engaging in these activities, biocontrol bacteria go through several regulatory processes at the transcriptional and post-transcriptional levels.

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Biological control of soil-borne pathogens by fluorescent pseudomonads

Biological control of soilborne pathogens by introduced microorganisms has been studied for over 65 years (9, 49), but during most of that time it has not been considered commercially feasible. Since about 1 965, however, interest and research in this area have increased steadily (9), as reflected by the number of books (10, 47,49, 152) and reviews about it (11,26,30, 106, 143, 153, 173, 174, 183) that have appeared . Concurrently, there has been a shift to the opinion that biological control can have an important role in agriculture in the future, and it is encouraging that several companies now have programs to develop biocontrol agents as commercial products. This renewed interest in biocontrol is in part a response to public concern about hazards associated with chemical pesticides. Microorganisms that can grow in the rhizosphere are ideal for use as biocontrol agents, since the rhizosphere provides the front-line defense for roots against attack by pathogens. Pathogens encounter antagonism from rhizosphere microorganisms before and during primary infection and also during secondary spread on the root. In some soils described as microbiologi­ cally suppressive to pathogens (172), microbial antagonism of the pathogen is especially great, leading to substantial disease control. Although pathogen­ suppressive soils are rare, those identified are excellent examples of the full potential of biological control of soilborne pathogens.

Biological control of soilborne plant pathogens in the rhizosphere with bacteria

Interest in biological control of plant pathogens has been stimulated in recent years by trends in agriculture towards greater sustainability and public concern about the use of hazardous pesticides There is now unequivocal evidence that antibiotics play a key role in the suppression of various soilborne plant pathogens by antagonistic microorganisms The significance of antibiotics in biocontrol, and more generally in microbial interactions, often has been questioned because of the indirect nature of the supporting evidence and the perceived constraints to antibiotic production in rhizosphere environments Reporter gene systems and bio-analytical techniques have clearly demonstrated that antibiotics are produced in the spermosphere and rhizosphere of a variety of host plants Several abiotic factors such as oxygen, temperature, specific carbon and nitrogen sources, and microelements have been identified to influence antibiotic production by bacteria biocontrol agents Among the biotic factors that may play a determinative role in antibiotic production are the plant host, the pathogen, the indigenous microflora, and the cell density of the producing strain This review presents recent advances in our understanding of antibiotic production by bacterial biocontrol agents and their role in microbial interactions

Antibiotic production by bacterial biocontrol agents

Certain strains of fluorescent pseudomonads are important biological components of agricultural soils that are suppressive to diseases caused by pathogenic fungi on crop plants. The biocontrol abilities of such strains depend essentially on aggressive root colonization, induction of systemic resistance in the plant, and the production of diffusible or volatile antifungal antibiotics. Evidence that these compounds are produced in situ is based on their chemical extraction from the rhizosphere and on the expression of antibiotic biosynthetic genes in the producer strains colonizing plant roots. Well-characterized antibiotics with biocontrol properties include phenazines, 2,4-diacetylphloroglucinol, pyoluteorin, pyrrolnitrin, lipopeptides, and hydrogen cyanide. In vitro, optimal production of these compounds occurs at high cell densities and during conditions of restricted growth, involving (i) a number of transcriptional regulators, which are mostly pathway-specific, and (ii) the GacS/GacA two-component system, which globally exerts a positive effect on the production of extracellular metabolites at a posttranscriptional level. Small untranslated RNAs have important roles in the GacS/GacA signal transduction pathway. One challenge in future biocontrol research involves development of new strategies to overcome the broad toxicity and lack of antifungal specificity displayed by most biocontrol antibiotics studied so far.

Regulation of antibiotic production in root-colonizing pseudomonas spp. and relevance for biological control of plant disease

Pseudomonas fluorescens 2-79 (NRRL B-15132) and its rifampin-resistant derivative 2-79RN10 are suppressive to take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. Strain 2-79 produces the antibiotic phenazine-1-carboxylate, which is active in vitro against G. graminis var. tritici and other fungal root pathogens. Mutants defective in phenazine synthesis (Phz-) were generated by Tn5 insertion and then compared with the parental strain to determine the importance of the antibiotic in take-all suppression on wheat roots. Six independent, prototrophic Phz- mutants were noninhibitory to G. graminis var. tritici in vitro and provided significantly less control of take-all than strain 2-79 on wheat seedlings. Antibiotic synthesis, fungal inhibition in vitro, and suppression of take-all on wheat were coordinately restored in two mutants complemented with cloned DNA from a 2-79 genomic library. These mutants contained Tn5 insertions in adjacent EcoRI fragments in the 2-79 genome, and the restriction maps of the region flanking the insertions and the complementary DNA were colinear. These results indicate that sequences required for phenazine production were present in the cloned DNA and support the importance of the phenazine antibiotic in disease suppression in the rhizosphere.

Role of a phenazine antibiotic from Pseudomonas fluorescens in biological control of Gaeumannomyces graminis var. tritici.

HOWELL, C. R., and R. D. STIPANOVIC. 1979. Control of Rhizoctonia solani on cotton seedlings with Pseudomonasfluorescens and with an antibiotic produced by the bacterium. Phytopathology 69:480-482. A strain of Pseudomonasfluorescens antagonistic to Rhizoctonia solani Alternaria sp., and Verticillium dahliae. A Fusarium sp. was only partially was isolated from the rhizosphere of cotton seedlings. An antibiotic inhibited and Pythium ultimum was unaffected. Treating cottonseed with strongly inhibitory to R. solani was isolated from P. fluorescens cultures P. fluorescens or pyrrolnitrin at time of planting in R. solani-infested soil and identified as pyrrolnitrin (3-chloro-4-[2'-nitro-3'-chlorophenyl]increased seedling survival from 30 to 79% and from 13 to 70%, respectively. pyrrole). The antibiotic also inhibited growth of other fungi associated with Pyrrolnitrin persisted for up to 30 days in moist 'nonsterile soil with no the cotton seedling disease complex including: Thielaviopsis basicola, measurable loss in activity. Additional key words: seed treatment, biological control. Fluorescent pseudomonads appear frequently among isolates portion was extracted twice with 100-ml volumes of ethyl ether. from plant rhizospheres (11) and often comprise an important The ether extracts were combined and taken to dryness in vacuo. component of the bacterial population (8,12). Pseudomonas spp. The residue was dissolved in chloroform at a concentration 1OOX were ineffective for control of damping-off by Rhizoctonia solani that in the original culture volume, and l-ml samples were streaked and Pythium spp. in nonsterile soil (3). However, Pseudomonas on thin-layer plates coated with silica gel 7GF. The plates were spp. have been used successfully to protect carnation cuttings developed in cyclohexane/chloroform/diethylamine (50:40:10, against Fusarium roseum f. sp. cerealis (9), potato seedpieces v/v) and observed under long and shortwave UV light to detect against certain pathogenic bacteria and fungi (4), and soybean fluorescing and adsorbing bands. Bands and blank areas were seedlings against Phytopthora megasperma var. sojae (13). scraped separately from the plates and eluted with acetone. In our work on seedling diseases of cotton we have isolated Acetone eluates diluted 1:10 with sterile water were assayed for fluorescent pseudomonads from the seedling rhizosphere and activity against R. solani by placing 100-/t1 aliquots in 7 mm report here: the isolation and identification of a fluorescent diameter wells cut in PDA plates inoculated simultaneously on the pseudomonad antagonistic to R. solani; the isolation and surface with agar plugs of the fungus. After 2 days of incubation the identification of an antibiotic produced by the isolate; and the plates were examined for mycelium-free zones around the wells. efficacy of that isolate or its antibiotic as a seed treatment to The eluate that contained active material again was streaked on prevent damping-off of cotton seedlings by R. solani. silica gel 7GF plates and developed with chloroform and the procedure for locating bands and detecting zones with antibiotic MATERIALS AND METHODS activity then was repeated. The eluate from the band showing inhibitory activity was concentrated in hot cyclohexane, and the Isolation and identification of an antagonistic bacterium from antibiotic was crystallized from it after cooling. Mass and NMR rhizosphere soil. Dilutions (10-6 and l0 , w/v) of rhizosphere soil spectra of the purified crystaline antibiotic were made and from 7-day-old cotton seedlings were spread in 0. l-ml samples on compared with standard spectra for authentic pyrrolnitrin plates of medium 523 in agar (7). Plates were incubated at 24 C for 4 obtained from the Fujisawa Pharmaceutical Co., Osaka, Japan. days then inoculated on the surface with PDA plugs of R. solani Antifungal spectrum of the antibiotic. Purified antibiotic (strain W-18 from cotton). After 3 days of incubation at 24 C the dissolved in 10% aqueous acetone was diluted to 25 vg/ml with plates were examined for bacterial colonies antagonistic to the sterile water. The solution then was assayed (by the methods growth of R. solani. Colonies with putative antibiotic activity were described previously for the chromatogram eluates) against the isolated and challenged with the fungus after 3 days. The most following fungi that have been associated with cotton seedling active isolate, a fluorescent pseudomonad, was subjected to diseases: Thielaviopsis basicola, Alternaria sp., Vertici/lium standard staining (10) and physiological tests (I), and identified to dahliae, Fusarium sp., and Pythium ultimum. species according to Bergey's manual (2). Efficacy of cottonseed treatment with bacterial cultures or Isolation and identification of an antibiotic from the antibiotic to control damping-off. Nonsterile soil containing 33 g of antagonistic bacterium. Plates containing medium 523 in agar were R. solani inoculum per 9 kg of soil was prepared and its level of spread with a bacterial suspension, incubated for 10 days at 24 C, infestation was determined according to methods described cut into 1-cm squares, and extracted with 200 ml of 80% aqueous previously (5). Dilutions of purified antibiotic in acetone were acetone for each 10 plates of culture medium. The extracts were prepared at 0, 20, 40, 60, 80, 100,200, and 400 pg/ml. One-milliliter filtered through cheesecloth and centrifuged at 12,000 g for 10 min samples were added to 0.2-g lots of diatomaceous earth carrier, and to remove particulate matter. The supernatant fluids were the acetone was removed in vacuo. Each of the 0.2-g samples then combined and condensed in vacuo at 40 C. Five grams of NaC1 was mixed with methyl cellulose, and coated onto 30 cottonseed per were added to each 100 ml of the aqueous concentrate and each sample, and assayed for optimum protective effect against R. solani This article is in the public domain and not copyrightable. It may be freely by the soil tube method described previously (5). reprinted with customary crediting of the source. The American PhytopathCottonseed treated with 200 /Ag/ml antibiotic were planted in ological Society, 1979. lots of 30 seed per flat in R. solani-infested or noninfested

C. R. Howell

Papers

Control of rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens and with an antibiotic produced by the bacterium.