The siderophore-producing bacterium, Bacillus subtilis CAS15, has a biocontrol effect on Fusarium wilt and promotes the growth of pepper

Abstract

In this study, the Chrome Azurol Sulphonate (CAS) agar plate assay for siderophores was used to screen bacteria from the rhizospheric soil of pepper in Hainan, China. As a result, Bacillus subtilis CAS15, which produced a large orange halo, was obtained. A dual-culture test showed that CAS15 strongly antagonized the growth of 15 plant fungal pathogens, with rates of inhibition ranging from 19.26 to 94.07%. Analysis of the siderophore production by the ESI-MS and DHB(G) assays showed that CAS15 produced the catecholic siderophore 2,3-dihydroxybenzoate-glycine-threonine trimeric ester bacillibactin, and that siderophore productivity was inhibited by iron. A pot culture experiment was used to study the effects of B. subtilis CAS15 on pathogen development and plant growth. CAS15 reduced the incidence of Fusarium wilt in pepper significantly, by 12.5–56.9%, which indicated that B. subtilis CAS15 induced systemic resistance to Fusarium wilt in pepper. Iron supplementation reduced this biocontrol effect. There were significant differences in plant height at 14, 21, 28 and 40 days, with treated plants growing 27.24–54.53% taller than controls. B. subtilis CAS15 also enhanced the yield of pepper by shortening the time to 50 percent flowering to 17.26 days, increasing the average fruit weight 36.92%, and increasing the average yield per plant 49.68%. This research showed that B. subtilis CAS15 has great potential for plant growth promotion and biological control.

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.