Infection of Corn Ears by Fusarium spp. Induces the Emission of

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Infection of Corn Ears by Fusarium spp. Induces the Emission of Volatile Sesquiterpenes Eva-Maria Becker,† Cornelia Herrfurth,‡ Sandra Irmisch,§ Tobias G. Köllner,§ Ivo Feussner,‡ Petr Karlovsky,† and Richard Splivallo*,† †

Molecular Phytopathology and Mycotoxin Research, Georg-August-University, Grisebachstrasse 6, 37077 Goettingen, Germany Department of Plant Biochemistry, Albrecht-von-Haller-Institute for Plant Sciences, Georg-August-University, Justus-von-Liebig-Weg 11, 37077 Goettingen, Germany § Max Planck Institute for Chemical Ecology, Hans-Knöll Straße 8, 07745 Jena, Germany ‡

S Supporting Information *

ABSTRACT: Infection of corn (Zea mays L.) ears with fungal pathogens of the Fusarium genus might result in yield losses and in the accumulation of mycotoxins. The aim of this study was to investigate whether volatile profiles could be used to identify Fusarium-infected corn ears. The volatiles released by corn ears infected by Fusarium graminearum, Fusarium verticillioides, and Fusarium subglutinans were studied. Volatile emission was recorded at 24 days postinoculation (dpi) and in a time series (from 4 to 24 dpi). Twenty-two volatiles were differentially emitted from Fusarium-infected versus healthy corn ears. These included C6− C8 compounds and sesquiterpenoids. All volatiles indicative of Fusarium infection were detectable as early as 4−8 dpi and continued to be produced to the final sampling time (early milk maturity stage). The induced emission of β-macrocarpene and βbisabolene correlated with an increased transcript accumulation of corn terpene synthase 6/11 (tps6/11). Additionally, the modification of volatile profiles after Fusarium infection was accompanied by the induction of plant defense compounds such as zealexins and oxylipins. Together, these results reveal a broad metabolic response of the plant to pathogen attack. Volatile biomarkers of Fusarium infection are promising indicators for the early detection of fungal infection before disease symptoms become visible. KEYWORDS: Fusarium spp., corn (Zea mays L.), volatile organic compounds, zealexins, oxylipins

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INTRODUCTION Worldwide production of corn reached 883 million tons in 2011, making corn one of the most important grain crops (faostat3.fao.org/). Fungal infection of corn can result in considerable economic losses due to yield reductions and the contamination of grain with toxic metabolites.1 Moreover, residues of infected corn plants serve as the inoculum sources for the infection of the subsequent crop.2 The fungal pathogens of corn include species of Fusarium, which typically cause ear infection (ear rot or ear mold) and which contaminate grains with mycotoxins (i.e., trichothecenes, fumonisins, zearalenone, and moniliformin) that are toxic to mammals.3 Fusarium infections in crop plants have been detected by mycological examination and by PCR.4 These techniques are laboratory based and require time- and labor-intensive sample preparation. Detection of volatile organic compounds (VOC) induced by fungal infection in planta was suggested as a promising method to monitor fungal infection.5 The analysis can potentially be performed directly in the field; volatile organic compounds (VOCs) can readily diffuse through plant tissues, and consequently their detection does not require their extraction from samples. VOCs might be induced in plants by interactions with other organisms (i.e., by attacking pathogens or insects) and by abiotic stresses such as drought or temperature stress.6,7 Volatile emission upon infection by fungi and insect pests has been documented from corn leaves and roots but not from the © 2014 American Chemical Society

most agriculturally relevant part of the corn, which is the ear. Herbivory and fungal infections of corn leaves or roots result in the emission of numerous terpenes and fatty acid-derived volatiles,8,9 some of which might be involved in direct or indirect plant defense. When corn is attacked by herbivores, for example, the plant synthesizes (E)-β-caryophyllene from farnesyl diphosphate to attract natural enemies of the herbivores.10,11 Other corn sesquiterpenes (SQTs) such as βbisabolene and β-macrocarpene, two volatile terpenes induced by herbivory or fungal infection and synthesized in corn by terpene synthase 6 (TPS6) and terpene synthase 11 (TPS11),12 are readily converted in corn to zealexins, which are nonvolatile acidic sesquiterpenoids involved in plant defense against pathogenic fungi and insect pests.13 Our aim here was to investigate the volatiles emitted by corn ears upon infection with toxicogenic Fusarium species. Volatiles were profiled from two corn lines infected with several strains of four Fusarium species, and the profiles from infected corn ears were compared with those from uninfected corn ears. The concentrations of relevant volatile infection markers were linked to factors such as plant development, fungal biomass, Received: Revised: Accepted: Published: 5226

February 10, 2014 May 6, 2014 May 11, 2014 May 11, 2014 dx.doi.org/10.1021/jf500560f | J. Agric. Food Chem. 2014, 62, 5226−5236

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Table 1. Overview of Fusarium Species and Strains Used To Inoculate Hybrid Corn and Dwarf Corn, Timing of Sample Collection, and Variables Measureda

and quantities of mycotoxins and plant defense compounds in plant tissue (i.e., zealexins, fatty acid-derived oxylipins).

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MATERIALS AND METHODS

hybrid maize

Chemicals. Authentic standards for 3-hexen-1-ol, heptan-2-ol, 1octen-3-ol, octan-3-one, and octan-3-ol were purchased from SigmaAldrich, Munich, Germany. β-Macrocarpene and β-bisabolene were produced using recombinant TPS6. The enzyme was heterologously expressed in Escherichia coli, purified, and incubated with the substrate farnesyl pyrophosphate. Enzyme products were collected from the headspace of the assay using SPME. Heterologous expression, enzyme assays, and product analysis were carried out as described earlier.12 αSelinene and β-selinene were identified using celery (Apium graveolens) seed oil (Carl Roth, Karlsruhe, Germany).14 Plant Material and Growth Conditions. Experiments were performed on two corn varieties, including the commercial hybrid variety Ronaldinio (obtained from KWS Saat AG, Einbeck, Germany) and the dwarf corn variety GaspeFlint landrace (originally collected in Quebec, Canada, provided by Prof. Dr. J. Schirawski, RWTH Aachen University, Germany). Corn was grown under greenhouse conditions at 26 ± 5 °C with a 16 h photoperiod (6600 lx at ear height) and a relative humidity of 58%. Corn seeds were sown in a soil mix consisting of 30% sand (1.00%) volatile infection markers detected in Fusarium-infected corn ears (β-macrocarpene and β-bisabolene) appear to be minor or undetectable infection markers in corn leaves, stems, and roots,11,36 where they might be fully transformed to nonvolatile zealexins involved in plant defense.36 Other major infection markers included the sesquiterpenoids α- and β-selinene, which are known components of celery oil.14 Both compounds have been reported to be involved in defense against pathogens and insects in plants other than corn37,38 and might have a similar role in corn. Infection Markers Are Detectable before Infection Symptoms Appear on Dehusked Corn. A second experiment was performed to investigate how quickly volatile infection markers were detectable after inoculation. This experiment was performed by sampling F. graminearum (strain FG2)- or F. verticillioides (strain FV1)-infected ears every 4 days, from 4 to 24 dpi. The earliest time point of 4 dpi was selected because at this time almost no infection symptoms are evident on ears that have not been dehusked. SPME/GC-MS analysis revealed that the majority of infection markers identified at 24 dpi (Table 2) were already detectable between 4 and 8 dpi (see Figure 3 for selected markers and the Supporting Information (Figure S2) for a heatmap of all markers). The induction of the major SQT markers βmacrocarpene and β-bisabolene was clearly the strongest with the most aggressive Fusarium species (F. graminearum, Figures 3 and 4A). Some volatiles, such as heptan-2-ol and (+)-cycloisosativene, decreased in concentration over time (i.e., after infection with F. graminearum), whereas others, such as octan3-one, β-selinene, β-macrocarpene, and β-bisabolene, increased over time and reached a maximum at 16−24 dpi (Figure 3 and Supporting Information Figure S2). Because most volatile markers were detectable at a very early stage of infection, they could be used for the early, nondestructive detection of fungal infection in the field (patent application, University of Goettingen, Germany, DE 101012204237.7). Sensitive portable devices such as ion mobility spectrometers (IMS) could be used for detection of infected ears in the field or during postharvest processes (i.e., storage, transport, or feed production). In the field, distinguishing healthy from infected corn plants might help guide decisions concerning the application of fungicides. In postharvest processes, infected corn ears might be separated from healthy ones and used for applications where mycotoxins are not a concern (i.e., for the production of biofuels). Sesquiterpenoid Infection Markers Are Also Detecatble from Corn Ears on Live Plants. SPME and OLS were used to capture the volatiles emitted by corn ears. SPME was performed on cut and frozen ears, whereas OLS was carried out

Figure 4. Disease severity and quantity of fungal DNA in hybrid corn ears inoculated with water (Cont), F. graminearum (strains FG1 and FG2), F. verticillioides (strains FV1 and FV2), or F. subglutinans (strain FSUB). Values are means + SE for two replicates for FSUB biomass and for theee to four replicates for all other treatments. The asterisk indicates statistical differences from control treatment (p < 0.05, Mann−Whitney U test).

Table 3. Myxotoxins in Fusarium-Infected Hybrid Corn Earsa mg toxin kg−1 dry weight ± standard error treatment control FG1 FG2 FV1 FV2 FSUB

DON

ZEN

>50b

0.55 ± 0.45

NIV

FB1

FB2

>10b 1.07 ± 0.45

>10b 0.51 ± 0.22

>50b

a Influence of inoculation with Fusarium strains on the formation of the mycotoxins deoxynivalenol (DON), zearalenone (ZEN), nivalenol (NIV), and fumonisins B1 and B2 (FB1, FB2) in hybrid corn ears at 24 dpi. bConcentration exeeds range of detection (trichothecenes, >50 mg kg−1; fumonisins, >10 mg kg−1).

a parent ion with m/z 204 and typical daughter ions having m/z values of 189 and 93 (Supporting Information Figure S1). On the basis of these properties, the six unidentified markers were tentatively assigned as sesquiterpenoids (SQTs). Sesquiterpenoids represented the major class of infection markers, and five sesquiterpenes, including β-selinene, αselinene, β-macrocarpene, β-bisabolene, and trichodiene, were dominant in the chromatograms (Figure 2). Trichodiene was emitted only by ears infected with FG1 and FG2, whereas βselinene, α-selinene, β-macrocarpene, and β-bisabolene were emitted by ears infected with all Fusarium species/strains tested. Healthy hybrid corn ears released high levels of 5232

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Figure 5. Disease severity and fatty acids in Fusarium-infected hybrid corn ears at 24 dpi. 13-HOD, (9Z,12Z)-8-hydroxy-9,12-octadecadienoic acid; 9-HOD, (10E,12Z)-9-hydroxy-10,12-octadecadienoic acid; 8-HOD, (9Z,12Z)-8-hydroxy-9,12-octadecadienoic acid; 13-HOT, (9Z,11E,15Z)-13hydroxy-9,11,15-octadecatrienoic acid; 9-HOT, (10E,12Z,15Z)-9-hydroxy-10,12,15- octadecatrienoic acid; 8-HOE, (9Z)-8-hydroxy-9-octadecenoic acid; 10-HOD, (8E,12Z)-10-hydroxy-8,12-octadecadienoic acid. The asterisk indicates statistical differences from control treatment (p < 0.05, Dunnet test). See Figure 4 caption for explanation of strain abbreviations. F.W. = fresh weight.

SPME samples might have released enzymes/substrates that would not colocalize in live plants. Despite these differences, both methods confirmed that sesquiterpenoids were major markers of Fusarium infection of corn ears (Table 2). Disease Severity Is Correlated with the Amount of Fungal DNA in Corn Ears. Disease severity, the percentage of the ears showing infection symptoms, was visually assessed for dehusked hybrid corn ears at the milk ripeness stage after inoculation with single Fusarium strains (Figure 4A). Strain FG1 was the most aggressive (>90% disease severity), FV2 and FSUB were the least aggressive (90%), whereas a slight yet not significant increase was detected in FV (disease severity 90%), an up-regulation in tps6/11 gene expression was also observed, but the expression of the housekeeping gene was reduced compared to uninfected plants. Possible explanations for this reduction are that RNA was degraded (i.e., due to fungal infection) or that a substantial proportion of the total RNA in the FG1 samples was of fungal origin. Both cases, however, should influence target genes and housekeeping genes at the same level. Overall, disease severity and expression levels of tps6/tps11 were correlated (R2 = 0.35, p = 0.006).

Figure 7. Gene expression and disease severity in dwarf maize ears at milk ripeness (14 dpi). Values are means ± SE for three to four replicate per treatment. The asterisk indicates difference from control treatment (p < 0.05, Mann−Whitney U test). Note that transcript levels of tps6/tps11 were normalized to the expression level of a housekeeping gene, which was stable in Cont, FA, and FV1 but which was reduced in FG. This reduction might reflect either RNA degradation (i.e., due to fungal infection) or the fact that an important share of the total RNA in the FG samples was of fungal origin.

in hybrid corn ears at 24 dpi is provided in Table 3. FG1 produced NIV and FG2 produced DON at concentrations >50 mg kg−1. Zearalenone (ZEN) was irregularly produced by F. graminearum strain 2 (FG2). Both F. verticillioides strains produced fumonisins B1 and B2, but FV1 produced much larger quantities of these mycotoxins than FV2 (Table 3). In time series with FG2 and FV1, DON was detectable at 4 dpi, whereas fumonisin FB1 was detectable only at 16 dpi (Supporting Information Figure S5). The relationship between emission of volatile markers and mycotoxin concentration over time is also presented in the Supporting Information (Figure S5). The relative quantity of trichodiene, which is the volatile precursor of trichothecenes, showed roughly the same pattern as DON production until 20 dpi (the correlation R24−20dpi = 0.43). The correlation became nonsignificant at 24 dpi (R24−24dpi = 0.02). The emission of octan-3-one, a common 5234

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fungal biomass quantitation by RT-PCR, RNA for gene expression analysis, and mycotoxins for quantitation by HPLC-MS. E.-M.B., S.I., and T.K. analyzed the gene expression of tps6/11. I.F. and C.H. quantitied oxylipins. E.-M.B. analyzed and processed all data. E.-M.B. and R.S. performed the statistics.

The up-regulation of zealexins A1 and A3 as well as of the terpene synthase genes tps6/11 indicates that β-macrocarpene and β-bisabolene, known to be fully transformed into nonvolatile zealexins in corn leaves, stems, and roots,12,36 are also partially transformed into zealexins in corn ears. However, the massive amounts of β-macrocarpene and β-bisabolene released by corn ears suggest that these volatiles might be further involved in direct plant defense or possibly in communication with neighboring plants (i.e., defense priming).41 Some of the fatty acid-derived volatiles (i.e., oxylipins42) identified in this study as infection marker volatiles are commonly produced by fungi and plants. Their source (fungal vs plant) can be determined by their different biosynthetic origins. Plants oxidize polyunsaturated fatty acids from membranes via the so-called lipoxygenase or oxylipin pathway, leading to the formation of green leaf volatiles such as 3-hexen1-ol via 13-H(P)OT as an intermediate (13-H(P)OT is the hydroperoxyl derivative of 13-HOT) (Figure 5).43 These green leaf volatiles (C6 volatiles) play a role in priming neighboring corn plants against insect attack.44 In addition, 13-H(P)OT may serve as a substrate for the formation of the phytohormone jasmonic acid, which is a master regulator of plant reaction against wounding and necrotrophic pathogens.45 In contrast, fungi oxidize the same substrates by a different enzyme family called psi-factor producing oxygenases.46 In fungi, 10-H(P)OD serves as the intermediate for the formation of C8 volatiles. Moreover, 8- and 10-H(P)OD regulate sexual reproduction and mycotoxin formation in ascomycetes.47,48 Indeed, the latter oxylipins were induced by Fusarium infection in the current study (Figure 5). Interestingly, the concept that oxylipins regulate mycotoxin formation is supported by the combined accumulation of the mycotoxin fumonisin B1 and octan-3one.49 Similarly, levels of thrichodiene, which is a known volatile precursor of thricothecenes in F. graminearum,50 were correlated with DON concentrations (Supporting Information Figure S5), again indicating a fungal origin. In summary, the volatile markers identified here might be used to discriminate healthy from Fusarium-infected corn ears. Unlike most existing techniques for detecting fungal infection in plants, volatile detection is nondestructive. If combined with the right technology, the markers identified in this study might be used for the management of corn fields and postharvest facilities.

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Funding

Funding for part of this research was provided by the German Federal Ministry of Education and Research (BMBF) within the framework of the project MykoSensExpert. Notes

The authors declare the following competing financial interest(s): EMB, RS and PK have applied for a patent describing the use of the volatile markers discovered in this study for identifying fungal infection in maize (patent application, University of Goettingen, Germany, DE 101012204237.7). The other author(s) declare that they have no competing interests.

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ACKNOWLEDGMENTS Katharina Döll and Philip Kössler are gratefully acknowledged for running the mycotoxin analysis on the HPLC-MS. We are thankful to J. Schirawski (RWTH Aachen University, Germany) for providing the seeds of dwarf-corn, to A. Prodi (University of Bologna, Italy) for Fusarium strain FV2, and to T. Miedaner (University of Hohenheim, Germany) for Fusarium strain FG1.

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ASSOCIATED CONTENT

S Supporting Information *

Figures S1-S5 and Tables S1 and S2. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*(R.S.) E-mail: [email protected]fl.ch. Fax: +49 551/391 2919. Phone: +49 551/393 3963. Author Contributions

P.K. wrote the proposal and secured funding. E.-M.B., R.S., and P.K. drafted the manuscript with input from all other coauthors. E.-M.B. handled all fungal and plant material, preformed corn ear infections, and monitored disease severity. E.-M.B., with guidance from R.S., carried out volatiles sampling by SPME, by OLS-GC/MS, zealexins extraction and quantitation and data analysis. E.-M.B., with guidance from P.K., extracted DNA for 5235

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dx.doi.org/10.1021/jf500560f | J. Agric. Food Chem. 2014, 62, 5226−5236