In planta reduction of maize seedling stalk lesions by the bacterial endophyte Bacillus mojavensis.

Maize (Zea mays L.) is susceptible to infection by Fusarium verticillioides through autoinfection and alloinfection, resulting in diseases and contamination of maize kernels with the fumonisin mycotoxins. Attempts at controlling this fungus are currently being done with biocontrol agents such as bacteria, and this includes bacterial endophytes, such as Bacillus mojavensis . In addition to producing fumonisins, which are phytotoxic and mycotoxic, F. verticillioides also produces fusaric acid, which acts both as a phytotoxin and as an antibiotic. The question now is Can B. mojavensis reduce lesion development in maize during the alloinfection process, simulated by internode injection of the fungus? Mutant strains of B. mojavensis that tolerate fusaric acid were used in a growth room study to determine the development of stalk lesions, indicative of maize seedling blight, by co-inoculations with a wild-type strain of F. verticillioides and with non-fusaric acid producing mutants of F. verticillioides. Lesions were measured on 14-day-old maize stalks consisting of treatment groups inoculated with and without mutants and wild-type strains of bacteria and fungi. The results indicate that the fusaric-acid-tolerant B. mojavensis mutant reduced stalk lesions, suggesting an in planta role for this substance as an antibiotic. Further, lesion development occurred in maize infected with F. verticillioides mutants that do not produce fusaric acid, indicating a role for other phytotoxins, such as the fumonisins. Thus, additional pathological components should be examined before strains of B. mojavensis can be identified as being effective as a biocontrol agent, particularly for the control of seedling disease of maize.

Is Quorum Signaling by Mycotoxins a New Risk-Mitigating Strategy for Bacterial Biocontrol of Fusarium verticillioides and Other Endophytic Fungal Species?

Bacterial endophytes are used as biocontrol organisms for plant pathogens such as the maize endophyte Fusarium verticillioides and its production of fumonisin mycotoxins. However, such applications are not always predictable and efficient. In this work, we hypothesize and review work that quorum sensing inhibitors are produced either by fungi or by pathogenic bacteria for competitive purposes, altering the efficiency of the biocontrol organisms. Recently, quorum sensing inhibitors have been isolated from several fungi, including Fusarium species, three of which are mycotoxins. Thus, we further postulate that other mycotoxins are inhibitors or quenching metabolites that prevent the protective abilities and activities of endophytic biocontrol bacteria within intercellular spaces. To test the aforementioned suppositions, we review work detailing the use of bioassay bacteria for several mycotoxins for quorum activity. We specifically focus on the quorum use of endophytic bacteria as biocontrols for mycotoxic fungal endophytes, such as the Fusarium species and the fumonisin mycotoxins.

Analyses of black Aspergillus species of peanut and maize for ochratoxins and fumonisins.

The genus Aspergillus section Nigri, or the black aspergilli, represents genetically closely related species that produce the mycotoxins, ochratoxins and the fumonisins. Fumonisin B1 (FB1) is of an added concern because it is also a virulence factor for maize. Our preliminary data indicated that black aspergilli could develop asymptomatic infections with maize and peanuts plants. Symptomless infections are potential problems, because under favorable conditions, there is a potential for accumulation of ochratoxins and the fumonisins in contaminated postharvest crops. In the present report, the ability of black aspergilli from peanuts and maize to produce ochratoxin A and FB1 on maize kernels was assessed. One hundred fifty strains from peanuts and maize were isolated from several southeastern and midwestern states. Aspergillus nigri (A. nigri var. nigri) was the dominant species (87%), while Aspergillus foetidus, Aspergillus japonicus, Aspergillus tubingensis, and Aspergillus carbonarius were infrequently isolated. None of the wild isolates produced detectable amounts of ochratoxins. However, we do report the occurrence of the fumonisins B1, B2, and B3. Of 54 field isolates, 30% (n = 16) produced FB1, 61% (n = 33) produced FB2, and 44% (n = 24) produced FB3. The amounts of fumonisins produced during the test period of 30 days suggest that these strains might be weak to moderate producers of fumonisin on maize. To our knowledge, this is a first report of FB1 and FB3 production by isolates of black aspergilli from an American cereal and legume.

The black Aspergillus species of maize and peanuts and their potential for mycotoxin production.

The black spored fungi of the subgenera Circumdata, the section Nigri (=Aspergillus niger group) is reviewed relative to their production of mycotoxins and their effects on plants as pathogens. Molecular methods have revealed more than 18 cryptic species, of which several have been characterized as potential mycotoxin producers. Others are defined as benign relative to their ability to produce mycotoxins. However, these characterizations are based on in vitro culture and toxins production. Several can produce the ochratoxins that are toxic to livestock, poultry, and humans. The black aspergilli produce rots of grapes, maize, and numerous other fruits and grain and they are generally viewed as post-harvest pathogens. Data are review to suggest that black aspergilli, as so many others, are symptomless endophytes. These fungi and their mycotoxins contaminate several major grains, foodstuffs, and products made from them such as wine, and coffee. Evidence is presented that the black aspergilli are producers of other classes of mycotoxins such as the fumonisins, which are known carcinogenic and known prior investigations as being produced by the Fusarium species. Three species are identified in U.S. maize and peanuts as symptomless endophytes, which suggests the potential for concern as pathogens and as food safety hazards.

Isolation and characterization of leu7-surfactin from the endophytic bacterium Bacillus mojavensis RRC 101, a biocontrol agent for Fusarium verticillioides.

Bacillus mojavensis is an endophytic bacterium patented for control of fungal diseases in maize and other plants. Culture extracts and filtrates from this bacterium were antagonistic to the pathogenic and mycotoxic fungus Fusarium verticillioides. However, the identity of the inhibitory substance from extracts of this bacterium has not been determined. An HPLC-MS analysis of the culture filtrate showed a major ion peak that was identified as a cyclic lipopeptide. Furthermore, collisional ion dissociation (CID) analysis indicated that this lipopeptide was surfactin, a cyclic heptapeptide linked to a ß-hydroxy fatty acid. A CID analysis of the peptide moiety was established by deduction and indicated that the peptide sequence consisted of two acidic amino acids and five hydrophobic amino acids with a sequence of Leu-Leu-Asp-Val-Leu-Leu-Glu. These spectra indicated that this bacterium produced Leu(7)-surfactin, which was toxic to F. verticillioides. Production of this cyclic lipopeptide is a characteristic of several species of Bacillus, but this is the first report of this very powerful biosurfactant from this endophytic species.

Interactions of Bacillus mojavensis and Fusarium verticillioides with a benzoxazolinone (BOA) and its transformation product, APO.

The benzoxazolinones, specifically benzoxazolin-2(3H)-one (BOA), are important transformation products of the benzoxazinones that can serve as allelochemicals providing resistance to maize from pathogenic bacteria, fungi, and insects. However, maize pathogens such as Fusarium verticillioides are capable of detoxifying the benzoxazolinones to 2-aminophenol (AP), which is converted to the less toxic N-(2-hydroxyphenyl) malonamic acid (HPMA) and 2-acetamidophenol (HPAA). As biocontrol strategies that utilize a species of endophytic bacterium, Bacillus mojavensis, are considered efficacious as a control of this Fusarium species, the in vitro transformation and effects of BOA on growth of this bacterium was examined relative to its interaction with strains of F. verticillioides. The results showed that a red pigment was produced and accumulated only on BOA-amended media when wild type and the progeny of genetic crosses of F. verticillioides are cultured in the presence of the bacterium. The pigment was identified as 2-amino-3H-phenoxazin-3-one (APO), which is a stable product. The results indicate that the bacterium interacts with the fungus preventing the usual transformation of AP to the nontoxic HPMA, resulting in the accumulation of higher amounts of APO than when the fungus is cultured alone. APO is highly toxic to F. verticillioides and other organisms. Thus, an enhanced biocontrol is suggested by this in vitro study.