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1 Introduction
The overuse of chemical pesticides to cure or prevent plant diseases has caused soil pollution and had harmful effects on human beings. Accordingly, to reduce the use of these chemicals, one possibility is to utilize the activity of microorganisms. It is desirable to replace chemical pesticides with materials that possess the following three criteria: (i) high specificity against the targeted plant pathogens, (ii) easy degradability after effective usage, and (iii) low cost of mass production. Products produced biologically or the microbial cells themselves are called biological control (biocontrol) agents or biological pesticides if they fulfill these criteria. Under these circumstances, the use of bacteria has been investigated mainly because genetic and biochemical analyses and the mass production of bacteria or bacterial products are much easier than those of fungi, and thus the advance of bacterial control is expected to have great potential. As bacterial control agents, Agrobacterium, Pseudomonas, Bacillus, Alcaligenes, Streptomyces, and others have been reported. Different mechanisms are involved in the actions of these bacteria against plant pathogens, such as parasitism, cross protection, antibiosis, and competition (1). Among them, the antibiotic mechanism is to operate when the metabolic products (antibiotics) produced by one species inhibit or suppress the growth of another species. This is the main mechanism that B. subtilis isolated in this study plays as a biocontrol agent.
It is critical to note in relation to biocontrol that while inhibition of the growth of plant pathogens is observed in vitro, often no correlation is observed between inhibition in vitro and reduction of diseases in a test in a greenhouse or in a field. Therefore, a plant test is essential to confirm the effectiveness of biocontrol agents.
As pseudomonads already have a long history as a candidate of bacterial control agents, and some reviews and reports of intensive research have been published (1), brief comments on pseudomonads are given and mainly the use of Bacillus spp. are emphasized.
1.1 Biological Control of Plant Pathogens by Pseudomonas Species
Pseudomonas strains are some of the most active and dominant bacteria in the rhizosphere and have been intensively investigated as biocontrol agents. P. fluorescens, P. putida, and P. cepacia were the predominant focus of research for practical applications. The strains of Pseudomonas produce several kinds of antibiotics, such as pyrolnitrin, pyoluteorin, and phenezine-1-carboxylate, all of which are closely related to the suppression of plant diseases.
Some Pseudomonas strains were confirmed to suppress the activities of plant pathogens by the production of siderophore or pseudobactin. Siderophores are extracellular, low-molecular-weight compounds that have a high affinity for ferric ions and play a role in biocontrol.
A siderophore-producing P. fluorescens induced an increase in the emergence of cotton seedlings in a soil infested with Pythium ultimum, while siderophore-deficient mutants of P. fluorescens derived by Tn5 mutagenesis induced a decrease in the emergence of seedlings.
1.2 Biological Control of Plant Pathogens by Bacillus Species
The use of the gram-positive Bacillus species as a biocontrol agent has been relatively rare and has been studied less intensively than that of the gram-negative bacteria. Bacillus subtilis is mainly studied and occasionally B. megaterium, B. cereus, B. pumilus, and B. polymyxa. As Bacillus spp. have the characteristics of omnipresence in soils, high thermal tolerance, rapid growth in liquid culture, ready formation of resistant spores, and are considered to be a safe biological agent, their potential as biocontrol agents is considered to be high. However, evaluation of the bacteria has focused primarily on the degree of disease suppression2ā6. The population dynamics and mechanism of suppression to plant pathogens in soil by Bacillus spp. have not been extensively investigated.
1.3 Production of Antifungal Substances
B. subtilis produces several kinds of antibiotics, e.g., bacillomycin, iturin, mycosubtilin, bacilysin, fengycin, plipastatin, and mycobacillin. However, the bacteria require a suitable substrate for production of these materials in soil. Investigation of the control of Streptomyces scabies in potatoes by B. subtilis revealed that more antibiotic was produced when the bacteria were grown in a water extract of soybean (7). Furthermore, when this bacterium was added to soil, buildup of potato scab was prevented, presumably because the soybean substrate supported antibiotic production in soil by B. subtilis, thus enhancing its ability to control the specific disease. A root system includes a wide variety of different substances whose compositions vary with changes in the metabolic state of different parts of the root system. The changes can be influenced by outside effects on the plants. Once a root is invaded by a pathogen, significant changes in exudation occur. Such changes could convert the environment into one hostile to the biocontrol agent. Because antibiotic activity is so dependent on outside events, biocontrol through its effects is likely to be unsatisfactory and certainly unpredictable, unless strains of microorganisms can be manipulated to make the synthesis of antagonistic compounds less susceptible to changes in nutrient sources. In this book, Chapters 2 and 3 explain the production of antifungal substancesāiturin, surfactin, and plipastatināand some examples of plant test related with these substances in soil.
1.4 Production of Siderophores
2,3-Dihydroxybenzoyl-glycine (2,3-DHBG) is known as only one siderophore produced by the gram-positive B. subtilis(8, 9). Bacterial siderophores are also known to have functions to help plant growth such as supplying iron via an iron-chelating function called the plant-growth-promoting rhizobacteria (PGPR) effect, which eventually leads to a decrease in disease occurrence. The strain of B. subtilis isolated in this study showed a wide suppressive spectrum on plant pathogens by producing antibiotics and 2,3-DHBG (Chapter 5). Gram-negative Escherichia coli produces a siderophore, enterobactin; and the E. coli mutant of entD-encoding enterobactin was complemented for enterobactin production by the sfp 0 gene of B. subtilis, which differs from sfp (surfactin production gene) by five base substitutions and one base insertion that truncated a 224-amino-acid residue in sfp to a 165-amino-acid residue. Similar complementation was found in the regulatory gene lpa-14 for the antibiotics iturin A and surfactin derived from a potential bacterial control agent of B. subtilis. The finding that B. subtilis produced not only antibiotics that suppress plant pathogens but also siderophores, and the regulation of these products by the gene lpa-14 indicates the possibility of enhanced effectiveness of biocontrol by the manipulation of the gene (Chapter 5).
1.5 Genetic Analysis
Some antifungal peptides produced by Bacillus species are synthesized by multienzyme complexes via nonribosomal mechanisms, and the molecular organization of these peptide synthetases encoded by the bacterial operons has been well analyzed. However, no relation between the genetic information and the suppressive effect on plant pathogens in soil has been reported. One regulatory gene lpa-14, which is responsible for the production of antibiotics, was found to be closely involved in the bacterial suppressive function against a plant disease in soil (Chapter 5).
B. subtilis RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, the iturin A synthetase operon of RB14 was identified and cloned, and the sequence of this operon was determined. The iturin A operon is composed of four open reading frames: ituD, ituA, ituB, and ituC. Each frame was characterized, and the homology with each frame of mycosubtilin was clarified. A 42 kb region of the B. subtilis RB14 genome, which contains a complete 38 kb iturin A operon, was transferred to the genome of a non-iturin A producer...
