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.

Maize seedling blight induced by Fusarium verticillioides: accumulation of fumonisin B1 in leaves without colonization of the leaves.

Fusarium verticillioides produces fumonisin mycotoxins during the colonization of maize, and fumonisin B1 (FB1) production is necessary for manifestation of maize seedling blight disease. The objective of this study was to address FB1 mobility and accumulation in seedlings to determine if proximal infection by F. verticillioides is necessary for FB1 accumulation. Taking advantage of an aconidial mutant known to have limited capability for seedling infection, tissue and soil samples were analyzed to compare wild-type F. verticillioides against the mutant. Inoculation with either strain caused accumulation of FB1 in the first and second leaves, but the mutants were unable to colonize aerial tissues. FB1, FB2, and FB3 were detected in the soil and seedling roots, but only FB1 was detected in the leaves of any treatment. These data suggest root infection by F. verticillioides is necessary for accumulation of FB1 in leaves, but the mechanism for accumulation does not require colonization of the leaf.

Nomenclatural realignment of Neotyphodium species with genus Epicholë.

Nomenclatural rule changes in the International Code of Nomenclature for algae, fungi and plants, adopted at the 18th International Botanical Congress in Melbourne, Australia, in 2011, provide for a single name to be used for each fungal species. The anamorphs of Epichloë species have been classified in genus Neotyphodium, the form genus that also includes most asexual Epichloë descendants. A nomenclatural realignment of this monophyletic group into one genus would enhance a broader understanding of the relationships and common features of these grass endophytes. Based on the principle of priority of publication we propose to classify all members of this clade in the genus Epichloë. We have reexamined classification of several described Epichloë and Neotyphodium species and varieties and propose new combinations and states. In this treatment we have accepted 43 unique taxa in Epichloë, including distinct species, subspecies, and varieties. We exclude from Epichloë the two taxa Neotyphodium starrii, as nomen dubium, and Neotyphodium chilense, as an unrelated taxon.

Tall fescue endophyte effects on tolerance to water-deficit stress.

BACKGROUND: The endophytic fungus, Neotyphodium coenophialum, can enhance drought tolerance of its host grass, tall fescue. To investigate endophyte effects on plant responses to acute water deficit stress, we did comprehensive profiling of plant metabolite levels in both shoot and root tissues of genetically identical clone pairs of tall fescue with endophyte (E+) and without endophyte (E-) in response to direct water deficit stress. The E- clones were generated by treating E+ plants with fungicide and selectively propagating single tillers. In time course studies on the E+ and E- clones, water was withheld from 0 to 5 days, during which levels of free sugars, sugar alcohols, and amino acids were determined, as were levels of some major fungal metabolites. RESULTS: After 2-3 days of withholding water, survival and tillering of re-watered plants was significantly greater for E+ than E- clones. Within two to three days of withholding water, significant endophyte effects on metabolites manifested as higher levels of free glucose, fructose, trehalose, sugar alcohols, proline and glutamic acid in shoots and roots. The fungal metabolites, mannitol and loline alkaloids, also significantly increased with water deficit. CONCLUSIONS: Our results suggest that symbiotic N. coenophialum aids in survival and recovery of tall fescue plants from water deficit, and acts in part by inducing rapid accumulation of these compatible solutes soon after imposition of stress.

One fungus, one name: defining the genus Fusarium in a scientifically robust way that preserves longstanding use.

In this letter, we advocate recognizing the genus Fusarium as the sole name for a group that includes virtually all Fusarium species of importance in plant pathology, mycotoxicology, medicine, and basic research. This phylogenetically guided circumscription will free scientists from any obligation to use other genus names, including teleomorphs, for species nested within this clade, and preserve the application of the name Fusarium in the way it has been used for almost a century. Due to recent changes in the International Code of Nomenclature for algae, fungi, and plants, this is an urgent matter that requires community attention. The alternative is to break the longstanding concept of Fusarium into nine or more genera, and remove important taxa such as those in the F. solani species complex from the genus, a move we believe is unnecessary. Here we present taxonomic and nomenclatural proposals that will preserve established research connections and facilitate communication within and between research communities, and at the same time support strong scientific principles and good taxonomic practice.

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.

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.

Use of a rep-PCR system to predict species in the Aspergillus section Nigri.

The Aspergillus niger aggregate within the A. section Nigri is a group of black-spored aspergilli of great agro-economic importance whose well defined taxonomy has been elusive. Rep-PCR has become a rapid and cost-effective method for genotyping fungi and bacteria. In the present study, we evaluated the discriminatory power of a semi-automated rep-PCR barcoding system to distinguish morphotypic species and compare the results with the data obtained from ITS and partial calmodulin regions. For this purpose, 20 morphotyped black-spored Aspergillus species were used to create the A. section Nigri library in this barcoding system that served to identify 34 field isolates. A pair-wise similarity matrix was calculated using the cone-based Pearson correlation method and the dendrogram was generated by the unweighted pair group method with arithmetic mean (UPGMA), illustrating four different clustered groups: the uniseriate cluster (I), the Aspergillus carbonarius cluster (II), and. the two A. niger aggregate clusters (named III.A and III.B). Rep-PCR showed higher resolution than the ITS and the partial calmodulin gene analytical procedures. The data of the 34 unknown field isolates, collected from different locations in the United States, indicated that only 12% of the field isolates were >95% similar to one of the genotypes included in the A. section Nigri library. However, 64% of the field isolates matched genotypes with the reference library (similarity values >90%). Based on these results, this barcoding procedure has the potential for use as a reproducible tool for identifying the black-spored aspergilli.

Chemotaxis disruption in Pratylenchus scribneri by tall fescue root extracts and alkaloids.

Tall fescue (Festuca arundinacea) forms a symbiotic relationship with the clavicipitalean fungal endophyte Neotyphodium coenophialum. Endophyte-infected grass is tolerant to nematode, but the factors responsible are unknown. One objective of this work was to determine if root extracts of tall fescue effected chemoreceptor activity of Pratylenchus scribneri by using an in vitro chemoreception bioassay. Another objective was to determine if specific ergot alkaloids (ergovaline, ergotamine, a-ergocryptine, ergonovine), and loline alkaloids, all produced by the fungal endophyte, altered chemotaxis with this bioassay. Methanolic extract from roots altered chemotaxis activities in this nematode but only from roots of plants cultured 45 > or = d, which repelled nematodes. Extracts prepared from noninfected grasses were attractants. This assay indicated that the alkaloids were either repellents or attractants. N-formylloline was an attractant at concentrations of 20 microg/ml and lower, while at higher concentrations it was a repellent. Ergovaline, the major ergot alkaloid produced by the endophyte, was repellent at both high and low concentrations and caused complete death of the nematodes.

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.

A single extraction method for the analysis by liquid chromatography/tandem mass spectrometry of fumonisins and biomarkers of disrupted sphingolipid metabolism in tissues of maize seedlings.

The fungus Fusarium verticillioides is a pathogen of many plants and produces fumonisins. In addition to their well-studied animal toxicoses, these toxins contribute to the development of maize seedling disease in susceptible maize varieties. Fumonisin disruption of sphingolipid biosynthesis occurs during pathogenesis. An extraction method was developed for the simultaneous analysis of fumonisins B(1) (FB(1)), B(2) (FB(2)) and B(3) (FB(3)), free sphingoid bases and sphingoid base 1-phosphates in maize tissues by liquid chromatography/linear ion trap tandem mass spectrometry. The method involved a single extraction using 1:1 acetonitrile:water + 5% formic acid (1 ml per 10 mg tissue). Mean recoveries ranged from approximately 50 to 99 percent, and limits of detection ranged from 10 fg microl(-1) to 6900 fg microl(-1). To test the efficacy of the method, seeds of a susceptible maize line were inoculated with a pathogenic, fumonisin-producing strain of F. verticillioides. The seedlings were then harvested, and fumonisin content, as well as sphingoid bases and their 1-phosphates, were measured in the leaf and root tissues. Fumonisin accumulation was significantly greater in leaf one compared to leaves two and three. While FB(1), FB(2), and FB(3) were detected in root tissues, FB(1) was preferentially accumulated in leaf tissues. Accumulation of sphingoid bases and their 1-phosphates was evident in roots and leaves of seedlings grown from inoculated seed, with the level of accumulation being similar in leaves 1, 2 and 3. The method developed was effective, fast, and sensitive for use in simultaneously measuring fumonisin in tissues and their effects on sphingolipid metabolite biomarkers of disease. The method should be useful for screening maize cultivars for susceptibility to F. verticillioides-induced seedling diseases.

Electrotransformation of Bacillus mojavensis with fluorescent protein markers.

Gram-positive endophytic bacteria are difficult to transform. To study the interactions between Bacillus mojavensis and maize, a method was developed to transform the microbe by electroporation with three integration plasmids expressing green, cyan and yellow fluorescent proteins (GFP variants). GFP Transformations were verified by antibiotic selection, microscopy and amylase deficiency, based on incorporation of the plasmids through recombination with the amylase gene. Phenotypic expressions of endophytism and fungal antagonisms of the transformants were the same as wild type stains. This represents the first successful transformation of this endophytic species with GFP markers.

Estimated fumonisin exposure in Guatemala is greatest in consumers of lowland maize.

Fumonisin mycotoxins contaminate maize worldwide. Analysis of maize samples (n = 396) collected from fields in Guatemala from 2000 to 2003 found that lowland maize (<360 m) had significantly more fumonisin B1 than highland maize (>1200 m). For example, 78% of the lowland samples collected at harvest in 2002 contained >0.3 microg/g of fumonisin B1, whereas only 2% of the highland samples contained >0.3 microg/g. Maize from the 2002 crop collected from storage in the highlands just before the 2003 harvest contained significantly more fumonisin B1 compared with levels at harvest in 2002. All Fusarium-infected kernels analyzed from 9 random lowland locations in 2001 were infected with fumonisin-producing Fusarium verticillioides and no other Fusarium species, whereas in samples from the highlands, only 5% of the Fusarium-positive kernels were F. verticillioides. In 2005, maize samples (n = 236) from the 2004 crop were collected from local markets in 20 Departments across Guatemala. The analysis showed that maize from lowland locations was often highly contaminated with fumonisin and was frequently transported to and sold in highland markets. Thus, fumonisin exposure in the highlands will be greatest in groups that obtain their maize in the market place from commercial vendors. Based on a recall study and published consumption data, a preliminary assessment of daily intake of total fumonisins was estimated. Consumption of nixtamalized maize products made from >50% of the maize from commercial vendors in 2005 could result in exposure exceeding the recommended WHO provisional maximal tolerable daily intake.

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.

Fumonisin disruption of ceramide biosynthesis in maize roots and the effects on plant development and Fusarium verticillioides-induced seedling disease.

The fungus Fusarium verticillioides infects maize and produces fumonisins, inhibitors of ceramide synthase. Seeds of the cultivar Silver Queen were inoculated with fumonisin-producing or non-fumonisin-producing strains of F. verticillioides. Leaf lesion incidence and severity of effects on root and stalk growth were significantly correlated with fumonisin in roots and disruption of sphingolipid metabolism in roots. Uninoculated seeds grown in soil watered with solutions of fumonisin B1 exhibited above-ground symptoms indicative of F. verticillioides-induced seedling disease and dose-dependent reduction in root mass that was inversely correlated with fumonisin B1, sphingoid bases, and sphingoid base 1-phosphates in roots. There was also evidence of an adaptive response to disrupted sphingolipid metabolism in both the virulence and watering assays, suggesting induction of pathways responsible for metabolism of sphingoid base 1-phosphates after prolonged exposure. The results suggest that fumonisin, and its effects on sphingolipids, could contribute to all aspects of F. verticillioides maize seedling disease.

Fumonisin production and bioavailability to maize seedlings grown from seeds inoculated with Fusarium verticillioides and grown in natural soils.

The fungus Fusarium verticillioides infects maize and produces fumonisins. The purpose of this study was to determine the ability of F. verticillioides to produce fumonisins in synthetic and natural soils and their biological availability to maize roots. Maize seeds were inoculated with a pathogenic strain of F. verticillioides (MRC826) and planted in synthetic and three different natural soils. There were statistically significant reductions in stalk weight and root mass and increased leaf lesions in the MRC826-treated seedlings in all soil types. Fumonisins were detected in all of the soils of seedlings grown from MRC826-inoculated seeds. The fumonisin produced in the soils was biologically available to seedlings as demonstrated by the statistically significant elevation of free sphingoid bases and sphingoid base 1-phosphates in their roots. These results indicate that F. verticillioides produced fumonisins in the autoclaved synthetic and natural soils and that the fumonisin produced is biologically available on the basis of evidence of inhibition of ceramide synthase.

Toxic effects of fumonisin in mouse liver are independent of the peroxisome proliferator-activated receptor alpha.

Fumonisin mycotoxins occur worldwide in corn and corn-based foods. Fumonisin B1 (FB1) is a rodent liver carcinogen and suspected human carcinogen. It inhibits ceramide synthase and increases tissue sphinganine (Sa) and sphingosine (So) concentrations. Events linking disruption of sphingolipid metabolism and fumonisin toxicity are not fully understood; however, Sa and So were shown to bind mouse recombinant peroxisome proliferator-activated receptor alpha (PPARalpha) in vitro. To investigate the role of PPARalpha in fumonisin hepatotoxicity in vivo, wild-type (WT) and PPARalpha-null mice were fed control diets or diets containing 300 ppm FB1, Fusarium verticillioides culture material (CM) providing 300 ppm FB1, or 500 ppm of the peroxisome proliferator WY-14,643 (WY) for 1 week. WY-fed WT mice exhibited hepatomegaly, an effect not found in WY-fed PPARalpha-null mice, and WY did not change liver sphingoid base concentrations in either strain. Hepatotoxicity found in FB1- and CM-fed WT and PPARalpha-null mice was similar, qualitatively different from that found in WY-treated animals, and characterized by increased Sa concentration, apoptosis, and cell proliferation. Transcript profiling using oligonucleotide arrays showed that CM and FB1 elicited similar expression patterns of genes involved in cell proliferation, signal transduction, and glutathione metabolism that were different from that altered by WY. Real-time RT-PCR analysis of gene expression demonstrated PPARalpha-dependence of lipid metabolism gene expression in WY-treated mice, whereas PPARalpha-independent alterations of genes in lipid metabolism, and other categories, were found in CM- and FB1-fed mice. Together, these findings demonstrate that FB1- and CM-induced hepatotoxicity in mice does not require PPARalpha.