Original articleThe siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper
Research highlights
►Siderophore producing. ► Bacillus subtilis. ► Biocontrol and plant growth promotion effect. ► Pepper.
Introduction
Iron is a cofactor for essential cellular processes in nearly all microorganisms [4]. Despite its abundance in nature, iron is often a growth-limiting nutrient because of the low solubility of ferric iron under aerobic conditions and a neutral pH, which is far below the approximately 1 μM that most organisms require for optimal growth. Because the bioavailability of iron is extremely limited in most of the natural habitats, a great number of bacteria including important pathogens produce ferric iron-chelating compounds known as siderophores to gain access to various iron sources [46].
Siderophores produced by a microorganism can bind iron with high specificity and affinity, making the iron unavailable for other microorganisms, and thereby limiting their growth. This strategy may certainly be involved in the biological control of plant diseases [10]. Competition for iron by siderophore production has long been recognized as an important antagonistic trait found in many of the bacterial biocontrol agents against plant pathogens [8]. Therefore, siderophore-producing microorganisms may have promise as biological control agents.
Bacillus species are well known for their ability to control plant diseases through various mechanisms, including the production of secondary metabolites. Bacillus spp., especially Bacillus subtilis, Bacillus cereus, and Bacillus amyloliquefaciens, are effective for the control of plant diseases caused by soil borne, foliar, and postharvest fungal pathogens [1], [2], [12], [18].
Microorganisms that grow in the rhizosphere are ideal as biocontrol agents, since this region provides the first line of defense. In the soil, plant roots normally coexist with bacteria and fungi that may produce siderophores capable of sequestering the available soluble iron, which could interfere with plant growth and function. However, plant roots are sometimes capable of taking up ferric complexes of siderophores and using these as sources of iron [35]. Thus, siderophores may play an important role in the competition between microorganisms and may also act as growth promoters [32].
Pepper (Capsicum annuum L.) is one of the most important vegetable crops grown extensively throughout the world, especially in temperate countries [27]. Fusarium wilt of pepper (C. annuum L.) is caused by the soil borne fungus Fusarium oxysporum Schl. f.sp. capsici. It may result in a severe loss of pepper quality and quantity, and it persists indefinitely in most soils because of its ability to colonize the roots of a number of weeds and to produce resistant spore structures. Since this pathogen is difficult to control with traditional methods, such as controlled irrigation and other cultural methods, biocontrol could be a better alternative.
Biological control of Fusarium wilts caused by formae specials of F. oxysporum has long been studied because the current available control methods are either inefficient or difficult to apply [3]. Unfortunately, due to the complexity of the biotic and abiotic interactions that play a role in biological control, the effectiveness of biocontrol agents against soil borne pathogens have been variable [14].
The aims of the present study were to screen for the siderophore-producing in the rhizosphere of pepper, rubber trees and tomato plants grown in Hainan China using the universal chrome azurol sulphonate (CAS) agar plate assay [40], and to evaluate their potentials in the biological control of F. oxysporum Schl. f. sp. Capsici, the causal agent of Fusarium wilt of pepper (C. annuum L.) and in the promotion of pepper plant growth.
Section snippets
Screening of siderophore-producing bacteria
The rhizospheric soil surrounding peppers, rubber trees and tomato grown in Hainan, China was screened for siderophore-producing bacteria according to the universal CAS agar plate assay [40]. On CAS agar plates, siderophore-producing (Sid+) bacteria form colonies with an orange halo. This occurs because iron is removed from the original blue CAS–Fe (III) complex during siderophore production. Plates were evaluated for the formation of siderophore halos after 5 days of colony incubation at 30 °C.
The screening of siderophore-producing bacteria
To quickly and efficiently screen for siderophore-producing soil bacteria, we used the CAS agar plate assay. By observing the formation of the halo and its size, a series of siderophore-producing bacteria were screened from the soil. After culture and purification by streaking 3 or 4 times, 97 bacterial isolates were obtained. Clones producing orange halos were observed on the preliminary screening plates (Fig. 1-A and B). When spotted and streaked on the fresh CAS agar plates, they produced
Discussions
In the CAS agar plate assay, the ternary complex CAS/iron III/hexadecyltri-methylammonium bromide serves as an indicator. When a strong chelator such as a siderophore, removes the iron from the dye, its color turns from blue to purple or orange. When this CAS complex is incorporated into agar plates, halos around the colonies were formed, indicating the production of a siderophore [22], [30], [40], [41]. In this study, a series of siderophore-producing isolates were screened from rhizospheric
Acknowledgements
The research was supported by the National Key Technology R&D Program of China (no. 2008BAD92B03).
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