In planta reduction of maize seedling stalk lesions by the bacterial endophyteBacillus 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.

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PAC1, a pH-Regulatory Gene from Fusarium verticillioides

Applied and Environmental Microbiology ◽

10.1128/aem.69.9.5222-5227.2003 ◽

2003 ◽

Vol 69 (9) ◽

pp. 5222-5227 ◽

Cited By ~ 116

Author(s):

Joseph E. Flaherty ◽

Anna Maria Pirttilä ◽

Burton H. Bluhm ◽

Charles P. Woloshuk

Keyword(s):

Fusarium Verticillioides ◽

Regulatory Gene ◽

Synthetic Medium ◽

Transcriptional Regulators ◽

Acidic Ph ◽

Alkaline Ph ◽

Wild Type ◽

Ph Responsive ◽

Alkaline Conditions ◽

Maize Kernels

ABSTRACT Fumonisins are a group of mycotoxins that contaminate maize and cause leukoencephalomalacia in equine, pulmonary edema in swine, and promote cancer in mice. Fumonisin biosynthesis in Fusarium verticillioides is repressed by nitrogen and alkaline pH. We cloned a PACC-like gene (PAC1) from F. verticillioides. PACC genes encode the major transcriptional regulators of several pH-responsive pathways in other filamentous fungi. In Northern blot analyses, a PAC1 probe hybridized to a 2.2-kb transcript present in F. verticillioides grown at alkaline pH. A mutant of F. verticillioides with a disrupted PAC1 gene had severely impaired growth at alkaline pH. The mutant produced more fumonisin than the wild type when grown on maize kernels and in a synthetic medium buffered at an acidic pH, 4.5. The mutant, but not the wild type, also produced fumonisin B1 when mycelia were resuspended in medium buffered at an alkaline pH, 8.4. Transcription of FUM1, a gene involved in fumonisin biosynthesis, was correlated with fumonisin production. We conclude that PAC1 is required for growth at alkaline pH and that Pac1 may have a role as a repressor of fumonisin biosynthesis under alkaline conditions.

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Chlorinated Auxins—How Does Arabidopsis Thaliana Deal with Them?

International Journal of Molecular Sciences ◽

10.3390/ijms21072567 ◽

2020 ◽

Vol 21 (7) ◽

pp. 2567 ◽

Cited By ~ 2

Author(s):

Antje Walter ◽

Lorenzo Caputi ◽

Sarah O’Connor ◽

Karl-Heinz van Pée ◽

Jutta Ludwig-Müller

Keyword(s):

Arabidopsis Thaliana ◽

Acetic Acid ◽

Model Organism ◽

Wild Type ◽

In Planta ◽

Bacteria And Fungi ◽

Tryptophan Derivatives ◽

Indole 3 Acetic Acid ◽

Iaa Conjugates

Plant hormones have various functions in plants and play crucial roles in all developmental and differentiation stages. Auxins constitute one of the most important groups with the major representative indole-3-acetic acid (IAA). A halogenated derivate of IAA, 4-chloro-indole-3-acetic acid (4-Cl-IAA), has previously been identified in Pisum sativum and other legumes. While the enzymes responsible for the halogenation of compounds in bacteria and fungi are well studied, the metabolic pathways leading to the production of 4-Cl-IAA in plants, especially the halogenating reaction, are still unknown. Therefore, bacterial flavin-dependent tryptophan-halogenase genes were transformed into the model organism Arabidopsis thaliana. The type of chlorinated indole derivatives that could be expected was determined by incubating wild type A. thaliana with different Cl-tryptophan derivatives. We showed that, in addition to chlorinated IAA, chlorinated IAA conjugates were synthesized. Concomitantly, we found that an auxin conjugate synthetase (GH3.3 protein) from A. thaliana was able to convert chlorinated IAAs to amino acid conjugates in vitro. In addition, we showed that the production of halogenated tryptophan (Trp), indole-3-acetonitrile (IAN) and IAA is possible in transgenic A. thaliana in planta with the help of the bacterial halogenating enzymes. Furthermore, it was investigated if there is an effect (i) of exogenously applied Cl-IAA and Cl-Trp and (ii) of endogenously chlorinated substances on the growth phenotype of the plants.

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Fusarium verticillioides SGE1 Is Required for Full Virulence and Regulates Expression of Protein Effector and Secondary Metabolite Biosynthetic Genes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-13-0281-r ◽

2014 ◽

Vol 27 (8) ◽

pp. 809-823 ◽

Cited By ~ 26

Author(s):

Daren W. Brown ◽

Mark Busman ◽

Robert H. Proctor

Keyword(s):

Secondary Metabolism ◽

Fusarium Verticillioides ◽

Fusaric Acid ◽

Orthologous Gene ◽

Gene Clusters ◽

Effector Proteins ◽

Surface Proteins ◽

Regulation Of Transcription ◽

In Planta ◽

Food And Feed

The transition from one lifestyle to another in some fungi is initiated by a single orthologous gene, SGE1, that regulates markedly different genes in different fungi. Despite these differences, many of the regulated genes encode effector proteins or proteins involved in the synthesis of secondary metabolites (SM), both of which can contribute to pathogenicity. Fusarium verticillioides is both an endophyte and a pathogen of maize and can grow as a saprophyte on dead plant material. During growth on live maize plants, the fungus can synthesize a number of toxic SM, including fumonisins, fusarins, and fusaric acid, that can contaminate kernels and kernel-based food and feed. In this study, the role of F. verticillioides SGE1 in pathogenicity and secondary metabolism was examined by gene deletion analysis and transcriptomics. SGE1 is not required for vegetative growth or conidiation but is required for wild-type pathogenicity and affects synthesis of multiple SM, including fumonisins and fusarins. Induced expression of SGE1 enhanced or reduced expression of hundreds of genes, including numerous putative effector genes that could contribute to growth in planta; genes encoding cell surface proteins; gene clusters required for synthesis of fusarins, bikaverin, and an unknown metabolite; as well as the gene encoding the fumonisin cluster transcriptional activator. Together, our results indicate that SGE1 has a role in global regulation of transcription in F. verticillioides that impacts but is not absolutely required for secondary metabolism and pathogenicity on maize.

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Amylopectin Induces Fumonisin B1 Production by Fusarium verticillioides During Colonization of Maize Kernels

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-1333 ◽

2005 ◽

Vol 18 (12) ◽

pp. 1333-1339 ◽

Cited By ~ 101

Author(s):

B. H. Bluhm ◽

C. P. Woloshuk

Keyword(s):

Quantitative Polymerase Chain Reaction ◽

Fusarium Verticillioides ◽

Animal Health ◽

Fumonisin B1 ◽

Polymerase Chain Reaction Analysis ◽

Defined Medium ◽

Wild Type ◽

Fungal Genes ◽

High Amylose ◽

Maize Kernels

Fusarium verticillioides, a fungal pathogen of maize, produces fumonisin mycotoxins that adversely affect human and animal health. Basic questions remain unanswered regarding the interactions between the host plant and the fungus that lead to the accumulation of fumonisins in maize kernels. In this study, we evaluated the role of kernel endosperm composition in regulating fumonisin B1 (FB1) biosynthesis. We found that kernels lacking starch due to physiological immaturity did not accumulate FB1. Quantitative polymerase chain reaction analysis indicated that kernel development also affected the expression of fungal genes involved in FB1 biosynthesis, starch metabolism, and nitrogen regulation. A mutant strain of F. verticillioides with a disrupted α-amylase gene was impaired in its ability to produce FB1 on starchy kernels, and both the wild-type and mutant strains produced significantly less FB1 on a high-amylose kernel mutant of maize. When grown on a defined medium with amylose as the sole carbon source, the wild-type strain produced only trace amounts of FB1, but it produced large amounts of FB1 when grown on amylopectin or dextrin, a product of amylopectin hydrolysis. We conclude that amylopectin induces FB1 production in F. verticillioides. This study provides new insight regarding the interaction between the fungus and maize kernel during pathogenesis and highlights important areas that need further study.

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Fusaric acid, aFusarium verticillioidesmetabolite, antagonistic to the endophytic biocontrol bacteriumBacillus mojavensis

Canadian Journal of Botany ◽

10.1139/b04-067 ◽

2004 ◽

Vol 82 (7) ◽

pp. 878-885 ◽

Cited By ~ 34

Author(s):

C W Bacon ◽

D M Hinton ◽

J K Porter ◽

A E Glenn ◽

G Kuldau

Keyword(s):

Fusarium Moniliforme ◽

Biocontrol Agent ◽

Bacterial Colonization ◽

Fusarium Verticillioides ◽

Fusaric Acid ◽

Endophytic Bacterium ◽

Fungal Endophyte ◽

Plant Diseases ◽

Control Group ◽

In Planta

An endophytic bacterium, Bacillus mojavensis Roberts, Nakamura & Cohan, was patented as a nonpathogenic biocontrol for plant diseases. However, before this bacterium can be used as a biocontrol agent, it must be evaluated against homologous competing organisms, some of which are equally successful endophytes, such as species of Fusarium that are symptomless endophytes, especially on maize. Preliminary field trials using this bacterium as a biocontrol agent against production of the fumonisin mycotoxins caused by infection of maize with Fusarium verticillioides (Sacc.) Nirenberg (= Fusarium moniliforme Sheldon) was less than that observed with greenhouse studies. Fusarium verticillioides and other species produce fusaric acid. Fusaric acid at concentrations as low as 22 µmol/L accounted for a 41% reduction in CFU compared with the control group, while concentrations of 223 µmol/L and higher resulted in total toxicity to the bacterium. Mutants of F. verticillioides that produced low concentrations of fusaric acid did not affect the endophytic CFU of the bacterium in seedlings. These results suggest that fusaric acid accounted for the reduction of bacterial colonization and the resulting poor biocontrol activity and suggested its importance to the fungus is as an antibiotic, which assists in the in planta competition for the intercellular niche colonized by F. verticillioides during its endotrophic state.Key words: Fusarium moniliforme, Fusarium verticillioides, bacterial endophyte, fungal endophyte, fumonisin.

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HXK1 regulates carbon catabolism, sporulation, fumonisin B1 production and pathogenesis in Fusarium verticillioides

Microbiology ◽

10.1099/mic.0.052506-0 ◽

2011 ◽

Vol 157 (9) ◽

pp. 2658-2669 ◽

Cited By ~ 23

Author(s):

Hun Kim ◽

Jonathon E. Smith ◽

John B. Ridenour ◽

Charles P. Woloshuk ◽

Burton H. Bluhm

Keyword(s):

Carbon Source ◽

Regulatory Networks ◽

Sole Carbon Source ◽

Metabolic Response ◽

Fusarium Verticillioides ◽

Fumonisin B1 ◽

Genetic Regulatory Networks ◽

Wild Type ◽

Increased Sensitivity ◽

Maize Kernels

In Fusarium verticillioides, a ubiquitous pathogen of maize, virulence and mycotoxigenesis are regulated in response to the types and amounts of carbohydrates present in maize kernels. In this study, we investigated the role of a putative hexokinase-encoding gene (HXK1) in growth, development and pathogenesis. A deletion mutant (Δhxk1) of HXK1 was not able to grow when supplied with fructose as the sole carbon source, and growth was impaired when glucose, sucrose or maltotriose was provided. Additionally, the Δhxk1 mutant produced unusual swollen hyphae when provided with fructose, but not glucose, as the sole carbon source. Moreover, the Δhxk1 mutant was impaired in fructose uptake, although glucose uptake was unaffected. On maize kernels, the Δhxk1 mutant was substantially less virulent than the wild-type, but virulence on maize stalks was not impaired, possibly indicating a metabolic response to tissue-specific differences in plant carbohydrate content. Finally, disruption of HXK1 had a pronounced effect on fungal metabolites produced during colonization of maize kernels; the Δhxk1 mutant produced approximately 50 % less trehalose and 80 % less fumonisin B1 (FB1) than the wild-type. The reduction in trehalose biosynthesis likely explains observations of increased sensitivity to osmotic stress in the Δhxk1 mutant. In summary, this study links early events in carbohydrate sensing and glycolysis to virulence and secondary metabolism in F. verticillioides, and thus provides a new foothold from which the genetic regulatory networks that underlie pathogenesis and mycotoxigenesis can be unravelled and defined.

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The Dent Stage of Maize Kernels Is the Most Conducive for Fumonisin Biosynthesis under Field Conditions

Applied and Environmental Microbiology ◽

10.1128/aem.05216-11 ◽

2011 ◽

Vol 77 (23) ◽

pp. 8382-8390 ◽

Cited By ~ 24

Author(s):

Adeline Picot ◽

Christian Barreau ◽

Laëtitia Pinson-Gadais ◽

François Piraux ◽

Daniel Caron ◽

...

Keyword(s):

Time Course ◽

Fusarium Verticillioides ◽

Animal Health ◽

Maximum Level ◽

Field Conditions ◽

In Planta ◽

Content Type ◽

Short Period ◽

Maize Kernels

ABSTRACTThe fungal pathogenFusarium verticillioidesinfects maize ears and produces fumonisins, known for their adverse effects on human and animal health. Basic questions remain unanswered regarding the kernel stage(s) associated with fumonisin biosynthesis and the kernel components involved in fumonisin regulation duringF. verticillioides-maize interaction under field conditions. In this 2-year field study, the time course ofF. verticillioidesgrowth and fumonisin accumulation in developing maize kernels, along with the variations in kernel pH and amylopectin content, were monitored using relevant and accurate analytical tools. In all experiments, the most significant increase in fumonisin accumulation or in fumonisin productivity (i.e., fumonisin production per unit of fungus) was shown to occur within a very short period of time, between 22/32 and 42 days after inoculation and corresponding to the dent stage. This stage was also characterized by acidification in the kernel pH and a maximum level of amylopectin content. Our data clearly support published results based onin vitroexperiments suggesting that the physiological stages of the maize kernel play a major role in regulating fumonisin production. Here we have validated this result forin plantaand field conditions, and we demonstrate that under such conditions the dent stage is the most conducive for fumonisin accumulation.

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Regulation of Fumonisin Biosynthesis in Fusarium verticillioides by a Zinc Binuclear Cluster-Type Gene, ZFR1

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2653-2659.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2653-2659 ◽

Cited By ~ 58

Author(s):

Joseph E. Flaherty ◽

Charles P. Woloshuk

Keyword(s):

Expressed Sequence Tag ◽

Sequence Similarity ◽

Fusarium Verticillioides ◽

Regulatory Gene ◽

Normal Growth ◽

Wild Type ◽

Binuclear Cluster ◽

Primary And Secondary Metabolism ◽

Type Gene ◽

Maize Kernels

ABSTRACT Fusarium verticillioides, a pathogen of maize, produces a class of mycotoxins called fumonisins in infected kernels. In this study, a candidate regulatory gene, ZFR1, was identified in an expressed sequence tag library enriched for transcripts expressed by F. verticillioides during fumonisin B1 (FB1) biosynthesis. ZFR1 deletion mutants exhibited normal growth and development on maize kernels, but fumonisin production was reduced to less than 10% of that of the wild-type strain. ZFR1 encodes a putative protein of 705 amino acids with sequence similarity to the Zn(II)2Cys6 binuclear cluster family that are regulators of both primary and secondary metabolism in fungi. Expression of ZFR1 in colonized germ and degermed kernel tissues correlated with FB1 levels. Overexpression of ZFR1 in zfr1 mutants restored FB1 production to wild-type levels; however, FB1 was not restored in an fcc1 (Fusarium C-type cyclin) mutant by overexpression of ZFR1. The results of this study indicate that ZFR1 is a positive regulator of FB1 biosynthesis in F. verticillioides and suggest that FCC1 is required for ZFR1 function.

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The Effect of Fusarium verticillioides Fumonisins on Fatty Acids, Sphingolipids, and Oxylipins in Maize Germlings

International Journal of Molecular Sciences ◽

10.3390/ijms22052435 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2435

Author(s):

Marzia Beccaccioli ◽

Manuel Salustri ◽

Valeria Scala ◽

Matteo Ludovici ◽

Andrea Cacciotti ◽

...

Keyword(s):

Fatty Acids ◽

Fusarium Verticillioides ◽

Fungal Growth ◽

Defense Responses ◽

Ceramide Synthase ◽

Ear Rot ◽

In Planta ◽

Disease Symptoms ◽

The Impact

Fusarium verticillioides causes multiple diseases of Zea mays (maize) including ear and seedling rots, contaminates seeds and seed products worldwide with toxic chemicals called fumonisins. The role of fumonisins in disease is unclear because, although they are not required for ear rot, they are required for seedling diseases. Disease symptoms may be due to the ability of fumonisins to inhibit ceramide synthase activity, the expected cause of lipids (fatty acids, oxylipins, and sphingolipids) alteration in infected plants. In this study, we explored the impact of fumonisins on fatty acid, oxylipin, and sphingolipid levels in planta and how these changes affect F. verticillioides growth in maize. The identity and levels of principal fatty acids, oxylipins, and over 50 sphingolipids were evaluated by chromatography followed by mass spectrometry in maize infected with an F. verticillioides fumonisin-producing wild-type strain and a fumonisin-deficient mutant, after different periods of growth. Plant hormones associated with defense responses, i.e., salicylic and jasmonic acid, were also evaluated. We suggest that fumonisins produced by F. verticillioides alter maize lipid metabolism, which help switch fungal growth from a relatively harmless endophyte to a destructive necrotroph.

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