WO2013135889A1

WO2013135889A1 - Volatile biomarkers for the detection of mycotoxin-producing fungal pathogens in maize plants

Info

Publication number: WO2013135889A1
Application number: PCT/EP2013/055460
Authority: WO
Inventors: Eva-Maria Becker; Petr KARLOVSKI; Richard Splivallo
Original assignee: Georg-August-Universität Göttingen, Stiftung Öffentlichen Rechts
Priority date: 2012-03-16
Filing date: 2013-03-15
Publication date: 2013-09-19
Also published as: DE102012204237A1

Abstract

The invention relates to a method for producing maize plants, maize cobs and/or maize kernels with reduced fungal infestation, in particular comprising the steps of testing the maize plants, maize cobs and/or maize kernels for at least two volatile organic substances from the group consisting of: longifolene, α-selinene, ß-selinene, a sesquiterpene with a Kovats index of 1442, a sesquiterpene with a Kovats index of 1445, ß-bisabolene and ß-macrocarpene, wherein the presence of the volatile organic substances indicates a fungal infestation, and treating the fungal infestation or separating out infested maize plants, maize cobs and/or maize kernels. The invention additionally relates to a method for identifying fungal infestation in maize plants, maize cobs and/or maize kernels. The invention further relates to a corresponding system and use thereof for testing maize plants, maize cobs and/or maize kernels for volatile organic substances (VOSs), and to the use of a calibration solution.

Description

Volatile biomarker for the detection of mycotoxin-producing fungal pathogens

corn plants

The present invention relates to a process for producing maize plants, maize cobs and / or corn kernels with reduced fungal attack, in particular comprising the step of testing the maize plants, corn cobs and / or corn kernels for at least two volatile organic compounds (VOCs) from the group consisting of: longifols , α-selenins, β-seleins, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolites and β-macrocarps, wherein the presence of the VOCs indicates a fungal attack, and the step of treating the fungal infestation or sorting out affected corn plants, corn cobs and / or corn kernels. In addition, the invention relates to a method for the detection of fungal infestation in maize plants, maize cobs and / or corn kernels. Furthermore, the invention relates to a corresponding system and its use for testing maize plants, maize cobs and / or corn kernels on VOCs, and to the use of a calibration solution for testing.

State of the art

Fungi can not only damage crops and reduce yields, but also produce toxins that then enter the food chain. So Fusarium ssp. Fumonisins as well as trichothecenes such as deoxynivalenol (DON), nivalenol (NIV) or T2 toxin. It is known that trichothecenes have multiple toxic effects on physiological processes within the human and animal body, for example they affect the immune system. In agriculture, fungal infection of crops leads to the widespread application of fungicides. This is associated with high costs and environmental impact.

Corn is the most important crop, with more than 810 million tons of production in 2009. In 2011 alone, 144,000 square miles were planted in the US. Fusarium fungi mainly attack cereals (Demyttenaere et al., 2004), in particular the crops maize and wheat. By Fusarium-In eVtionen yield losses of up to 50% can result. In the US, the annual loss is estimated at up to $ 1 billion. In addition, in Germany alone, the annual use of fungicides in 2007 amounted to about 436 million euros. The sprays are applied on suspicion and with a quantity surcharge of approx. 30%. The appropriate use of fungicides would thus result in a high potential for savings. Thus, there is a need for methods and measuring systems that detect fungicides on plants and crops safely and in a timely manner.

With the exception of the visual inspection of cereal stocks by farmers and experts, there is currently no efficient system for the early detection of a field infection. The exact determination of fungal biomass by molecular biological methods, eg quantitative real-Tlme PCR (polymerase chain reaction) and the determination of toxin l Concentration with analytical methods, such as HPLC-MS

(High-performance liquid chromatography-mass spectrometry) are time-consuming, cost-intensive and unsuitable as a quick decision-making basis for farmers.

As mentioned above, in addition to the yield reduction in the agricultural industry, the problem is that mushroom-infested foods are consumed. If fungal infestation of food is not detected in time and the affected food is not sorted out, the toxins produced by the fungus remain in the food cycle and are consumed by humans. In the event that the fungal infection is detected in time, the infested plants or plant fruits can be sorted out, so they no longer get into the food circulation. However, they can then still be used, for example, for the production of fuels. Due to the resulting lower cost but the fight against the fungus in the cultivation of plants is beneficial. The early detection of fungal attack on crops would thus be advantageous to take timely countermeasures, for example, to treat the plants with suitable sprays.

An additional problem in the agricultural industry is the infestation of crops by other pests, such as pests (Piesik et al., 2011a / b). So far, different markers have already been described which can indicate the infestation of pests, for example insect pests or fungal infestation.

It is known that plants respond to pest infestation, such as fungus infestation or pest infestations, as well as in response to changing environmental conditions such as changes in temperature, ozone levels, waterlogging, etc., with the emission of VOCs. Here, different amounts and types of VOCs can be emitted, already knowing a large variety of different VOCs. VOCs are small hydrocarbons of a wide variety of compounds (e.g., aldehydes, ketones, esters) which have high volatility and can be transported over long distances (see, for example, Degenhardt et al., (2009)). VOCs are produced by a variety of organisms (from the plants themselves or even from the pests), and the composition of the volatile components for a species may be specific under the given environmental conditions and physiological conditions. F Fischer et al. (Ί999Υ).

The detection of VOCs can be carried out by GC (gas chromatography), GC-MS (gas chromatography coupled with mass spectrometry), IMS (ion mobility spectrometer) or EN (Electric Nose). The use of electronic noses to detect VOCs emitted by the fungi themselves is described in Magan et al. (2000).

With regard to infection with Fusarium spp. The volatile sesquiterpene trichodiene was identified as a suitable marker for the biosynthesis of trichothecenes. Trichodiene is a volatile starting material in the trichothecene pathway through the fungus itself, mycotoxins such as DON, DAS, NIV, etc., and has been known for many years (Jelen et al., 1997). This group cultivated different Fusarium spp. on autoclaved Wheat grains and analyzed them using Headspace GC-MS. Jelen et al. also describes that Fusarium phosphors can produce and emit β-bisabolene.

The VOC emissions of Fusarium cuimorum infection under field conditions was first reported by Perkowski et al. (2008). The group performed monitoring of infected wheat and triticale stocks using SPME (solid phase microextraction) GC / MS technique. In addition to trichodiene, no other relevant sesquiterpenes could be identified under these conditions.

Girotti et al. (2010) investigated VOC emissions of different Fusarium spp. On sterile rice grains and detected a wide range of unknown sesquiterpenes in addition to the trichodienes.

In addition, it is known that the volatile profile of in wiro experiments deviates significantly from the complex of volatile organic compounds in i / zVo experiments. In in vitro experiments, the occurrence and concentration of specific volatile organic compounds depend on the choice of medium (eg, sterile cereal grains) (described in Perkowski et al., 2008), but the killing of the plant material is lacking in such experiments biotic factors that can affect fungal growth, such as plant defense reactions, which is why the fungi can spread unhindered.

The article by Köllner et al. (2008) describes that ß-bisabolene is detectable in maize that is infested with insect pests. In addition, β-macrocarps were detected in the roots of maize plants. However, no β-macrocarp could be found in the leaves after pest infestation (see Figure 11, on page 20787). The same is also said in Koellner et al. (2004) reports. In addition, β-macrocarpene has been detected in plant tissue (Huffaker et al. (2011)). Huffaker et al. report that the VOC β macrocarpene is converted into related non-volatile compounds.

The dissertation by C. Zipfel (2007) investigates the emission of volatile organic compounds (VOCs) by transgenic and non-transgenic maize plants, especially after pest infestation, over longer periods of time. Table 2.15 on page 32 of the dissertation shows the emission of various VOCs, for example β-seleins, α-selines and β-bisabolene. However, no explanation for the emission of these VOCs could be given and various causes were mentioned, for example pest infestation, cultivation temperatures, ozone levels, waterlogging, herbicides etc. Possible fungal attack by Fusarium spp. was neither considered nor are the experiments carried out suitable for this purpose. In particular, a fungal attack is not always visually recognizable and other detection methods are therefore necessary to detect or exclude fungal infection.

The dissertation by P. Thakeow (2008) describes the emission of VOCs from wood to fungal infestation.

In view of the findings to date, there is still a great need for suitable measuring systems and methods for detecting fungal infestation in maize plants. Methods for detecting pests on plants are already known in the prior art. Thus, the mirror 50/2010, 13. 12. 2010, describes a sensor of

Ackerschädlinge "smells".

In addition, it is known that maize plants can withstand stress conditions, i. after injury, infection or fungal infection, produce various volatile organic substances and release them to the environment. In Schmelz et al. 2003. "Simultaneous analysis of phytohormones, phytotoxins, and volatile organic compounds in plants", PNAS, 100, 10552 -10557, describes a GC-MS based analysis of volatile organic compounds in plants. Huffaker et al. "Novel Acidic Sesquiterpenoids Constitute a Dominant Class of Pathogen-Induced Phytoalexins in Maize", Plant Physiology (2011), Vol. 156, 2082-2097, relates to ubiquitous sesquiterpenoid phytoalexins in maize plants. Demyttenaere et al. 2004 "Use of headspace solid-phase microextraction and headspace sorptive extraction for the detection of the volatile metabolites produced by toxigenic Fusarium species", Journal of Chromatography A 1027 (1 2), 147-154, relates to the detection of toxic Fussarium spp. using Headspace Sorptive Extraction (HSSE). Tholl et al. "Practical approaches to plant volatile analysis", The Plant Journal (2006) 45, 540-560 describes various methods for the determination of volatile organic substances in plants. Piesik et al. 2011 "Cereal crop volatile organic compound induction after mechanical injury beetle herbivory (Ou / ema spp.), Or fungal infection (Fusarium spp.)", Journal of Plant Physiology 168 (9), 878-886 describes the induction of volatile organic matter in plants infected by pests. Koellner et al. "Protonation of a Neutral (S) -β-Bisabolene Intermediate Is Involved in (5) -β Macrocarpene Formation by the Maize Sesquiterpene Synthases TPS6 and TPS11", Journal of Biological Chemistry, Vol. 283, No. 30, pp. 20779-20788 the formation of β-macrocarcene in plants by the synthases TPS6 and TPS11.

Koellner et al. 2004 "The sesquiterpene hydrocarbons of maize (Zea mays) form five groups with distinct developmental and organ-specific distributions", Phytochemistry, 65, 1895 -1902 relates to "sesquiterpene chemistry" in maize.

FR 2650475 AI, DE 19950396 C2, DE 10002880 Cl, and WO 02/32222 AI disclose methods for determining the condition of plants that are attacked by, for example, pests. Here, for example, radiothermometric or thermographic examination or optical and chemical sensors are used.

In particular, there is a need for measurement systems and methods for early, reliable, inexpensive, and easy implementation. In addition, there is a need for methods that can produce and detect maize plants and corncobs / corn kernels with reduced fungal infestation. There is also a need for methods that allow the targeted use of sprays, or the sorting of corn plants / maize / corn kernels with fungal infestation.

Summary of the invention

As described above, the emission of VOCs in plants depends on a variety of factors, such as pest infestation, fungal infestation, cultivation temperatures, ozone in the air, waterlogging, etc. In order to make a reliable statement regarding the causes of the emission of VOCs was in the context of the present invention developed a trial system that allowed to largely control the relevant environmental influences on plants. In particular, the plants were cultivated under "greenhouse conditions", since in the field, not all environmental influences can be controlled and can not be considered in retrospect. In the context of the present invention, a genetic engineering method was used for the exact analysis of fungal infestation (polymerase chain reaction, PCR, of fungal DNA). Only in this way could a clear statement be made whether the measured VOCs were measured on fungus-infected or healthy maize plants. After the markers according to the invention have now been found, the pest infestation can now be clearly detected independently of external influences in the field.

As in Koellner et al. (see above), could not be detected in the leaves of corn ß macrocarpene after pest infestation. Huffaker et al. have also reported (see above) that β-macrocarpene is converted to acidic, non-volatile compounds, possibly explaining that no β-macrocarpene is released from the maize leaves.

It was therefore surprising that β-macrocarpene could be detected after fungal attack in maize plants with corncobs or in corn plants from full bloom. Thus, the present invention is based inter alia on the surprising finding that the VOC emission is different in maize with corncob and without corncob. Without this knowledge, it has not been possible to distinguish whether a particular VOC emission is caused in maize plants due to the growth stage, or caused by (temporary) pest infestation or the like (see above-mentioned dissertation by C. Zipfel).

Using the present invention, in the experiments carried out, fungal infestation could already occur at a very early stage after infection, e.g. with Fusarium spp. (For example, from 4 days after inoculation with a spore suspension) are detected. At this time, no external symptoms (for example, burns around the plungers) can be seen on the outside. After harvesting the flasks and removing the leaflets (at 4 days after inoculation dpi), only 2 out of 6 flasks showed a weak infestation. Of course it depends on the concentration of

Spores, the specific characteristics of the fungi strains used and the growth conditions, how fast the pathogens can develop on the plant. Under natural conditions of infection in the field can be detected without destructive measures, an infestation with the human eye probably much later.

The markers (VOCs) according to the invention thus enable a safe and early detection of fungal infestation in maize plants with corn cobs or, respectively, from full bloom. The same applies to the examination of maize cobs which have been separated from the plant, ie harvested or separated for analysis, and maize kernels. Only the realization that certain VOCs can be used as markers for fungal attack when corn cobs develop, enabled the present invention, in particular, the knowledge is decisive for the timing of the measurement of the marker, ie it is measured when corn cobs develop, and for the suitable measuring points, ie near the corncobs. In particular, an early fungal infestation can optionally be detected with the method and system according to the invention, since the markers have a high sensitivity and specificity and, in addition, because the knowledge about the plant parts (corncob) which emit the VOCs, a targeted measurement of already very low VOCs Concentrations allowed.

Furthermore, the present invention makes it possible to provide a suitable measuring system which, on the basis of the findings described here, with regard to its constructional properties and / or with respect to its measuring functions, is adapted for the detection of fungal infestation in maize plants with corn cobs and can be used from the plant separated corncobs and corn kernels. In particular, it is advantageously possible to use GC, GC-MS, IMS, FAIMS or cheaper sensor expert systems (eg electrochemical sensor arrays, colorimetric sensors, and electrical / electronic noses), as the combination of markers according to the invention will test for a characteristic VOC Fingerprint, with the measurement systems calibrated to this VOC fingerprint.

In the context of the present invention, it has additionally been unexpectedly found that mixed infections can lead to certain VOCs which are suitable for the detection of individual strains no longer being detectable in a mixed infection of two strains. For the mixed infection variants tested different strains were used, which were also tested as a single infection. The strains used in the experiments each have different properties (e.g., lineage, mycotoxin spectrum, aggressiveness). Grown on the plant, the two species are in a competitive relationship, in which usually one of the species gets the upper hand. Depending on the nature of this complicated interaction, synthesis / emission or even suppression of certain VOCs signals occurs. As shown in FIGS. 2 and 3, mixed infections of Fusarium graminearum (strain 1) with Fusarium verticillioides (strain 1) (MIX 1 in FIGS. 2 and 3) and Fusarium graminearum (strain 2) with Fusarium verticillioides (strain 2) (MIX 2 in Figures 2 and 3) and tested the suitability of the VOCs as markers in the mixtures. Due to the influence of the

Fungal strains with each other, it was only possible to find suitable markers by the different strains have been studied individually and in mixtures.

It has been shown that ß-seleins is particularly suitable for the detection of MIX1 and MIX2 infections. In addition, β-selenin is characteristic of both Fusarium graminearum strains and Fusarium verticillioides strain 1. In addition, in order to be able to detect an infestation with Fusarium verticillioides strain 2, β-macrocarpene can preferably additionally be tested.

In a preferred embodiment, the method according to the invention is therefore possibly suitable for detecting mixed infections.

One embodiment of the invention therefore relates to a method for producing maize plants (with corncobs), maize cobs and / or corn kernels with reduced fungal attack, comprising the steps: (i) providing maize plants, corn cobs and / or corn kernels, the maize plants having reached at least the stage of growth BBCH coding 65,

(ii) testing the maize plants of step (i), corn cobs and / or maize kernels for at least two volatile organic compounds from the group consisting of: longifolene, a-selinin, beta-selenin, a sesquiterpene with a Kovats index of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolene and β-macrocarpene, wherein the presence of the volatile organic substances indicates a fungal attack,

(iii) treating the corn plants from step (ii) with fungal infestation with spray (s) effective against the fungus found in step (ii) or separating maize plants, corn cobs and / or corn kernels from step (ii ) with fungal infestation of those without fungal infestation, and

(iv) recovering corn plants, corn cobs and / or corn kernels from step (iii) with reduced fungal attack compared to the maize plants, corncobs and / or corn kernels provided in step (i).

Another embodiment relates to a method of detecting fungal infestation in maize plants, corn cobs and / or corn kernels, comprising testing the maize plants, corn cobs and / or corn kernels according to the invention as described in step (ii) as described herein, wherein the presence of the volatile organic materials indicates fungal infestation , The term "presence of volatile organic compounds" means that the VOCs are detectable, i. that a measurement signal is recognizable that is stronger than a possible background noise.

Another embodiment relates to a system for testing corn plants, corn cobs and / or corn kernels for volatile organic compounds (VOCs), the system comprising a meter comprising at least two volatile organic compounds selected from the group consisting of: longifolene, alpha-selenin , β-selenin, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolene and β-macrocarpenes.

Another aspect of the invention relates to the use of the system of the invention for testing maize plants, corn cobs and / or corn kernels for volatile organic compounds (VOCs) for detecting fungal infestation.

Another aspect is the use of a calibration solution comprising at least two organics from the group consisting of: longifolene, α-selenin, β-selenin, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolene and β-macrocarpenes, for calibrating a meter for testing maize plants, corn cobs and / or corn kernels for volatile organics for detecting fungal infestation.

Description of the figures

In the context of the present invention, the following abbreviations are used: SQT = sesquiterpene; KI = Kovatsindex; AI calculated / calculated = calculated Kovats Retention Index; Dpi = days after inoculation; Fusarium gr. = Fusarium graminearum; FG1 = Fusarium graminearum strain 1; FG2 = Fusarium graminearum strain 2; Fusarium sub. = Fusarium subglutinans; FSUB = Fusarium subglutinans strain 1; Fusarium vert. - Fusarium verticillioides; FV1 = Fusarium verticillioides strain 1; FV2 = Fusarium verticillioides strain 2; MIX1 = mixed inoculation with FG1 and FV1; MIX2 = mixed inoculation with FG2 and FV2; SQT unknown = unknown sesquiterpene; no ID = no identification possible = unknown.

Figure 1: The figure shows a listing of the molecular masses of VOCs. Explanations:

¹ the signals hexanal, 2-heptanone and 2-pentylfuran have been included in the table as constitutive VOCs for maize. They are not statistically relevant.

² the identification took place with the help of the databases (NIST 08, Adams and Wiley mass spectral libraries) and the comparison with the literature.

³ the identification was carried out by direct gas chromatographic comparison with a standard.

Figure 2: Shown is a table based on normalization of mean integrated peak areas (n = 4) (raw data), where 1 = maximum peak area within the signal and 0 = VOC signal is absent. This illustration clearly shows the differences / ratios for the individual treatments within a volatile signal.

The "italic and bold" labeled signals hexanal, 2-heptanone and 2-pentylfuran have been included as constitutive VOCs for maize in the table. They are not statistically relevant. Figure 2 contains no statistical analysis. The values for the integrated peak areas were averaged for each selected marker within the treatments (FG1, FG2, control ...) and normalized by the maximum value.

Color coding of the cells: white = 0 = the signal does not appear in the corresponding treatment; light gray = <0.75 = the signal appears moderately in the corresponding treatment; dark gray = the signal appears strongly in the corresponding treatment (the maximum value is marked with 1).

FIG. 3: This figure is based on statistical evaluation, Kruskal-Wallis test (Siegel & Calstellan (1988), p <0.01.

The symbol: "//" means that the VOCs are present only in infected samples. The symbol "/" indicates that the VOCs are present in the control but overexpressed in infected plants. Gray areas mean that the VOC is at 4-2 dpi. The symbol: "\\" means that the VOCs were only in control (healthy plants) but were absent during treatment, equivalent to control. The symbol: "V means that the VOCs are present in the control and in infected plants but are under-expressed after infection. Detailed description of the invention

In the context of the invention, the term "maize plants" or "maize plants with corn cob" in connection with the fungus detection method according to the invention on corn plants from the growth stage BBCH (Federal Biological Research Center for Agriculture and Forestry, Bundessortenamt and Chemical Industry) coding 65. This growth stage draws by a male inflorescence with full bloom, ie upper and lower panicle branches in bloom and a female inflorescence with fully pushed scar tissue. The growth stage can be determined with the naked eye.

The fruit / corncob development starts with the main flower of the plant, which is characterized by the BBCH-65. The flower marks the beginning of the piston development or the plant-physiological transition from the vegetative phase to the generative phase. From this point on, the corn plants according to the invention are thus referred to as maize plants with corn cobs. More preferably, a piston is present as soon as the grain formation is visible to the naked eye.

In a preferred embodiment, the corn plants are used at the earliest 4 days from BBCH-65, more preferably at the earliest 5, at the earliest 6, at the earliest 7, at the earliest 8, at the earliest 9, or at the earliest 10 days after BBCH-65. It is likewise possible to carry out the test method according to the invention from the point in time at which the grain formation in the bulb is noticeable, palpable or discernible to the naked eye.

The present invention relates to a process for the production of maize plants, in particular maize plants having at least BBCH-65 growth stage, maize cobs and / or corn fungi with reduced fungal attack, comprising the steps (i) - (iv).

Step (i) is to provide maize plants, corncobs (separated from the maize plant), and / or corn kernels (separated from the maize plant / corncob).

The term "providing" includes culturing corn plants and optionally harvesting the corncobs and corn kernels. However, the maize plants and the maize cobs and corn kernels separated from the maize plant may also be produced separately and then subjected to the process according to the invention. If the method comprises cultivating the corn plants, the use of sprays can be controlled and controlled according to the invention. If the culturing and harvesting of the plants is done in a separate process, the sorting out of mushroom-infested maize plants, corn cobs and / or corn kernels can be controlled and controlled according to the invention. Preferably, Zea mays is used.

Preferably, the method in step (i) comprises at least one step selected from the group consisting of: cultivating corn plants, harvesting corncobs, and providing harvested corncobs or corn kernels.

The process can be carried out on maize plants, provided that the maize plants are already developing corn cobs. The procedure can be carried out before or after the harvest. Additionally preferably, the provision of the corn plants in the present method comprises cultivating maize plants in a substrate, for example in soil, in particular in the field or in agriculture. Particularly preferably, the corn plants are cultivated on easily heatable soils with good nutrient and water supply. Less preferred is cultivation in soil with waterlogging or heavy soils. In order to produce as natural a substrate as possible for the cultivation of the hybrid corn in the greenhouse, two-thirds commercial compost soil was mixed with 1/3 sand during the experiments. There was a moderate fertilizer intake with commercial fertilizer (urea, potash, phosphorus and micronutrients).

Step (ii) comprises testing the maize plants, corncobs and / or corn kernels for at least two, more preferably at least three, more preferably at least four, more preferably at least five, more preferably at least six and more preferably at least seven or exactly two, preferably exactly three, more preferably exactly four, more preferably exactly five, further preferably exactly six, more preferably exactly seven, further preferably exactly eight volatile organic substances from the group consisting of: longifols, α-selines, β-selines, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolene and β-macrocarpene, where the presence of volatile organic substances indicates a fungal attack.

In another embodiment, the group of volatile organic substances is formed from: longifoles, α-selines, β-selines, β-bisabolites and β-macrocarps. It is preferred to test for at least two, more preferably at least three, more preferably at least four, more preferably five or exactly two, preferably exactly three, more preferably exactly four, more preferably exactly five VOCs from the above group , In addition, preference is given in the process according to the invention to at least β-selenines and β-macrocarps, more preferably to at least β-selinen, β-

Macrocarps and α-selines tested. More preferably, not only two VOCs from the group consisting of alpha-selenin, SQT 1442 and SQT 1445 are tested.

More preferably, only VOCs that are not found in maize plants having at least BBCH-65 growth stage, corn cobs and / or corn kernels infested with fungi and other pests are tested. In particular, only VOCs which were not detectable in FIG. 2 in the column "healthy corncob VOCs" (white field) are preferably tested. Particularly preferred are only volatile organic substances from the group consisting of: Longifolen, α-selines, ß-seleins, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, ß-bisabolene and ß-macrocarpene tested. In addition, it is preferable that the volatile organic matters indicated by the symbol "w" and / or the volatile organic substances indicated by the symbol "\" in Fig. 3 are not used as a marker.

In another embodiment, β-selenin and β-macrocarpene, and additionally at least one or two of the aforementioned group of volatile organic compounds (VOCs) are tested, in addition at least longiferols and / or β-bisabolene / α-seleins are additionally tested. In another embodiment, only β-selenins and β-macrocarps and optionally α-seleins are tested. In principle, any number of volatile organic compounds (VOCs) can be tested, but preferably a maximum of 10, a maximum of 7, a maximum of 6, a maximum of 5 or a maximum of 4 volatile organic substances (VOCs). In particular, it is additionally preferred that only VOCs from the group consisting of: Longifolen, α-selins, ß-seleins, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, ß-bisabolene and ß-acrocarpen or aus the group consisting of: Longifolen, α-Selinen, ß-Selinen, ß-Bisabolen and ß-Macrocarpen be tested.

In one embodiment, in addition to the above-mentioned groups of VOCs, one, two, three or more organic volatiles selected from the group consisting of: 2-hexanone, 3-octanone, 3-octanol, sesquiterpene with a Kovats index of 1410, sesquiterpenes with a Kovatsindex of 1477, sesquiterpenes with a Kovatsindex of 1485, sesquiterpenes with a Kovatsindex of 1488, and sesquiterpenes with a Kovatsindex of 1547.

In a variant of the method, in step (ii), in addition to the above-mentioned groups of VOCs, it is also possible to test for volatile mycotoxins or mycotoxin precursors, preferably trichodens, or other suitable markers, in particular VOCs prepared by the particular pest itself.

The values for the Kovats index of VOCs given herein are calculated on the basis of alkane hydrocarbons. The method is described in Kovats, E. (1958).

Because of the relationships between pest infestation and VOC emission described herein (particularly as illustrated in the figures), the advantages of the various VOCs or groups of VOCs will be apparent to those skilled in the art and the VOCs may be selected according to the application desired. For example, the type of fungal attack to be tested (type of fungus, mixture of fungus species) can be taken into account. It is also possible to select specific VOCs depending on the type and sensitivity of the meter.

The test method according to the invention is preferably carried out from time BBCH-65, more preferably at least 4 days after BBCH-65, more preferably at the earliest 5, at the earliest 6, at the earliest 7, at the earliest 8, at the earliest 9, or at the earliest 10 days after BBCH-65. A corn stock in the field has reached BBCH-65 stage when approximately 50% of the plants are at this stage. In particular, it is possible to observe the plants in the center of the culture, whereas the plants can be disregarded at the field edges (for example up to 1, 2, 3, 4 or 5 meters from the edge of the field). It is likewise possible to carry out the test method according to the invention from the point in time when the grain formation in the bulb can be recognized by the naked eye or the grains can be felt.

In a preferred embodiment of the method, the fungal attack by Fusarium spp. caused, in particular by a fungus from the group consisting of: Fusarium graminearum, Fusarium subglutinans, Fusarium verticillioides, and mixtures thereof, preferably Fusarium graminearum and / or Fusarium verticillioides.

In another embodiment, fungal infestation is considered to be indicated when the volatile organics are measured in concentrations above specified thresholds. In this process, maize plants can first be treated with at least BBCH growth stage, corn cobs and / or maize kernels other than fungi and other pests for which the desired VOCs are tested. The threshold that indicates fungal infestation when exceeded is then chosen above the levels of corn plants, corn cobs and / or corn kernels not affected by fungi and other pests. For example, a threshold value of 10%, 30%, 50% or more above the measured value may be selected. For example, as a threshold value, a measured value of the magnitude of at least two or three times the strength of the signal of the measurement noise can be selected. For example, a threshold for an open-loop measurement over 24 hours may be 0.1-4.0 pg. However, the threshold value depends, for example, on the sensitivity of the measuring device and can therefore be suitably selected by the person skilled in the relevant system.

Additionally preferably, the step of testing for volatile organics comprises calibrating the meter by means of a sample containing the respective volatile species. Suitable mixing ratios of the VOCs can be readily selected by the skilled person. For example, the VOCs to which the meter responds less sensitively can be used in larger quantities. The meter may be calibrated before or possibly during or between the measurements. The fingerprint of the calibration solution is then considered characteristic of pest infestation.

For testing the volatile organic substances, for example, a measuring device may be used which is selected from the group consisting of: gas chromatographs (GC), gas chromatographs coupled with mass spectrometers (GC-MS), ion mobility spectrometers (IMS), high field ion mobility spectrometers with asymmetric waveform (FAIMS, high-field asymmetric waveform ion mobility spectrometry),

electrochemical sensors, electrochemical sensor arrays, colorimetric sensors, and electrical / electronic noses.

Volatile organics testing may be performed, for example, in the headspace surrounding the corncobs of the maize plants, corncobs separated from the plant (e.g., harvested), and / or corn kernels, preferably by head-space sampling. Most preferably, a dynamic headspace sampler is used. The term "head-space" is the (immediate) air space that surrounds the plant parts above the earth. The distance of the measuring sensors depends on the type and sensitivity of the sensor.

Basically, there are two methods for the collection of volatile organic components. A distinction is made here between the dynamic or static method. In dynamic headspace sampling, a stream of air is passed continuously over a sample (eg infected plant, fungal culture) located in a special vessel of inert material (eg glass). With this air flow, the VOCs emitted by the sample are passed through a glass cartridge equipped with a suitable filter (eg activated carbon). On it the components are enriched. The process usually takes several hours (Jelen et al., 1995). Subsequently, the absorbed volatile components are extracted from the filter material with organic solvents. In the static method (static headspace sampling), the sample is transferred into an airtight container, usually made of glass. By diffusion, the volatile components gradually dissolve away from the sample and accumulate in the surrounding air, also called headspace. Temperature and humidity play a crucial role in this process (Balasubramanian & Paniqrahi (2011)). The emitted VOCs can attach to an absorbent matrix which is exposed for a certain time to the ambient air of the sample. The most common form within static methods is solid phase microextraction (SPME). Here, a fiber coated with specific polymers is exposed to the headspace of any sample (this is described, for example, in Tholl et al., 2006). The absorption is selective according to the properties of the absorbent material.

The measurement method can be carried out in such a way that a gas chromatographic analysis is followed by the static or dynamic method of collecting volatile components.

In addition to the exact, but very time and cost intensive analysis by GC-MS (gas chromatography with mass spectrometry coupling) exist portable electronic sensors, so-called e-noses (electrical / electronic noses). These can also be used to detect specific VOCs. They are typically constructed as a multi-sensor array consisting of several chemoresistive metal oxide sensors (eg, In ₂ O ₃ , SnO ₂ ) (Presicce et al (2006), Abramson et al., 2005 ¹ ); Falasconi et al. (2005), Dickinson et al. (1998V).

Gobbi et al. (2011) and Falasconi et al. (2005) investigated the suitability of such electronic sensors for the prediction of fumonisin contamination in maize. They chose in vitro conditions for their investigations. With the help of the E-nose technique and GC / MS Olsson et al. (2002) Balasubramanian et al (2007) used E-nose for ergosterol-based classification within barley samples.

In measuring the VOCs according to the present invention, it is preferable to test the headspace in the vicinity of the corncobs of the maize plants, the corncobs cut off from the plant and / or the corn kernels, with the appropriate distance from the corncobs of the corn plants, the corncobs separated from the plant and / or corn kernels depends on the meter and can be selected by the skilled person. Preferably, a measuring device is used which has a resolution in the centimeter range and can distinguish between adjacent plants. Thus, for example, an affected plant can be distinguished from its neighboring uninfected plant.

For example, testing may be done by aspirating air from the environment of the corn plants, particularly from the vicinity of the corncobs of the plants, the corncob and / or the corn kernels, into the meter, which air is tested for volatile organics. The air is preferably measured in 0.7-1.8 m, preferably 1-1.5 m, height from the ground. The horizontal distance to the plant is for example 5 to 50 cm.

Step (iii) comprises treating corn plants having at least BBCH-65 growth stage with fungal infestation with spray (s) effective against the fungus found in step (ii), or separating corn plants, corn cobs and / or Corn kernels with fungal infestation of those without fungus. Testing in step (ii) results in a measurement that either indicates fungal attack or indicates that there is no fungal attack. Based on the measurement result, it is determined whether to act or whether only the next time for a measurement is determined. Action based on the measurement results may include treating the plants with antifungal agents, but also removing affected plants from the plant culture.

Preferably, treating the corn plants in step (iii) comprises applying sprays, preferably sprays, to Fusarium spp., More preferably to one or more fungi from the group consisting of Fusarium graminearum, Fusarium subglutinans, Fusarium verticillioides, and mixtures thereof. In particular, Fusarium infection is a buttock sinus. Preferably, the spray contains a fungicide or mixture of several fungicides.

With the method of the invention, spray can be selectively applied to positively tested (i.e., fungus-infested) maize crops. The areas of corn acreage that have no fungal attack can be left out.

If the corn plants, corn cobs or corn kernels have already been harvested, the action in step (iii) of the method according to the invention is based on the measurement results in the sorting out of affected corn plants, maize cobs or corn kernels. Thus, the

Fungus infestation based on a certain amount of maize plants, corncobs or maize kernels is reduced or maize plants, maize cobs and / or corn kernels with reduced fungal infestation are produced.

Additionally preferably, the separation of fungus-affected and unaffected maize plants, maize cobs and / or corn kernels takes place by means of an agricultural machine, preferably by means of a harvester.

In the method described herein, the corn plants, corn cobs (and corn) separated from the plant in step (i) may be provided in batches (ie, non-continuous) or in a continuous process, wherein the corn plants, corn cobs and / or corn kernels in Step (ii) be tested batchwise or in a continuous process, and

wherein the process step in (iii) comprises separating the affected and unaffected maize plants, corn cobs and / or corn kernels in different batches in which the respective volatile organic substances are detected / not detected, or at least detected in different amounts, or

the process step in (iii) comprises the continuous separation of fungal infested and non-infested maize plants, corn cobs and / or corn kernels.

In another embodiment of the invention, the provision of corn plants or maize plants with corncobs includes planting and cultivating maize plants to the full bloom or development of corncobs. The separation of maize plants with fungal infestation of those without fungal infestation can then be done by either harvesting only the corncobs with fungal infestation, or the corncobs without fungal infestation. Thus, the corncobs are harvested separately with fungus and those without fungus. The order here is arbitrary, but preferably the corncobs are harvested without fungal attack to prevent further spread of the fungus.

Devices and methods for sorting out corn plants, corn cobs and / or corn kernels are known to the person skilled in the art. For example, infested maize plants, corn cobs and / or corn kernels can be sorted out by hand, or shot with compressed air from the conveyor belt. In particular, batch sorting is preferred, with the sensor being held / integrated into the sump with the batch.

Based on the existing techniques, the devices of the invention can be provided without problems. For example, the corn plants, corn cobs and / or corn kernels can be transported on a conveyor belt. Above the conveyor belt is the measuring system according to the invention. If the characteristic VOCs are measured, a certain part of the conveyed material, which was under the measuring sensor at that moment, is considered as having been attacked by fungi. This part of the conveyed material can then be directed to a different transport path than that of the unaffected conveyed material and thus be separated.

If the maize plants, corn cobs and / or maize kernels are batched in containers, the measurement procedure can also be carried out in the respective containers, thus classifying them in unaffected and fungal batches.

The measuring systems according to the invention can be used in particular directly on harvesting machines, so that a reduction of the fungal load takes place during the harvesting process.

If the method according to the invention comprises the cultivation of maize plants, steps (ii) and (iii) can also be carried out repeatedly. For example, step (iii) may involve either treating or rejecting infested plants, or merely determining the next time to measure (step (ii)).

Step (iv) of the method according to the invention comprises harvesting maize plants, corn cobs and / or maize kernels with reduced fungus attack compared to the maize plants, corncobs and / or corn kernels provided in step (i).

In step (iv) the "product" of the process according to the invention is obtained. If a defined amount of maize plants, corncobs and / or corn kernels obtained is compared with the same, comparable amount of maize plants, corncobs and / or maize kernels provided in step (i), the fungal infestation in the product obtained is lower. If corn plants are cultivated in step (i), the product obtained has a reduced fungal attack compared to a product which would have been obtained if the process according to the invention, which may for example comprise the selective use of spraying agents, had not been carried out.

The invention also relates to a method for detecting fungal infestation in maize plants with at least BBCH-65 growth stage, (separated from the plant) corn cobs and / or Corn kernels comprising testing the corn plants, corn cobs and / or corn kernels according to step (ii) as described herein, wherein the presence of the volatile organic matter indicates fungal infestation. The method of detecting fungal infestation is performed to determine if fungal infestation is present. If there is no fungal attack, there is no need to act, that is, for example, no step of spraying or sorting out corn plants, corn cobs or corn kernels is necessary. If there is a fungal attack, action can be taken as described above. All of the preferred embodiments described for the method of producing maize plants, corn cobs, and / or corn kernels with reduced fungal infestation may also be used for the method of detecting fungal infestation, and vice versa.

Furthermore, the invention relates to a system for testing maize plants having at least BBCH-65 growth stage, corn cobs and / or corn kernels for volatile organic compounds (VOCs), the system comprising a measuring device comprising at least two, preferably three volatile organic substances from the Group consisting of: longifolene, α-selenin, β-selenin, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, ß-bisabolene and ß-macrocarpenes. All of the preferred embodiments described for the method of producing corn plants, corn cobs, and / or reduced fungal corn kernels may also be used for the system for testing corn plants, corn cobs, and / or corn kernels for volatile organics (VOCs), and vice versa.

Preferably, the system is mounted on an agricultural utility vehicle or on a drivable drone. For example, drones can inspect corn acreage independently of large agricultural vehicles to detect fungal infestation. Of course, a human-powered vehicle can equally be used for driving off and testing the crops. Likewise, the system may be configured to be worn by a human.

Additionally preferably, the system includes a computer with appropriate software that handles the control of the meter.

The system preferably comprises a control element which controls a sorting system for sorting maize plants, maize cobs and / or corn kernels using measurement signals which are transmitted from the measuring device to the control element.

In addition, the measuring device is preferably selected from the group consisting of: gas chromatographs (GC), gas chromatographs coupled with mass spectrometers (GC-MS), in particular field-portable GC-MSs such as Z-noses (available from Electronic Sensor Technology, USA), ion spectrometers. Mobility spectrometers (IMS), high field ion mobility spectrometers with asymmetric waveforms (FAIMS), electrochemical sensors, electrochemical sensor arrays, colorimetric sensors, and electrical / electronic noses.

Electric / electronic noses typically include three elements: a sensor array exposed to the VOCs, means that can convert the sensor signals into a readable format or signal, and software that processes the data can. Conventional sensors can be used according to the invention, for example metal oxide sensors (MOX), eg tin oxide or zinc oxide, MOSFET sensors (metal oxide semiconductor field effect sensors), conductive polymer sensors, infrared sensors, quartz sensors (QMB, quartz Crystal Micro Balance ), Field effect sensors, or fiber optic sensors.

The other measuring devices described above, for example IMS, GC-MS, can also be used according to the invention by adapting commercially available devices according to the invention. "According to the invention" means here that the measuring devices are calibrated according to the invention, equipped with a unit containing the calibration solution according to the invention, or geometrically mounted in the system so that the sampling can be done according to the invention in the vicinity of corn cobs or corn kernels. The operation of an IMS is described for example in DE102009011102 AI.

Additionally preferably, the system comprises a means that converts the measurement signal into a visually or audibly perceptible signal if fungal infestation is detected. Such a means may be, for example, a lamp, a display or a speaker. Upon perception of the signal can then be responded to the fungal attack accordingly.

In a further preferred embodiment, the system comprises a calibration unit containing a calibration solution, wherein the calibration solution contains a mixture of VOCs according to the invention and the system can automatically access this calibration solution to automatically carry out calibration processes at suitable intervals.

Additionally preferably, the system includes a control element that controls a device for delivering spray to corn plants using measurement signals communicated from the gauge to the control element.

The invention also relates to a calibration solution comprising at least two, preferably at least three, organic substances from the group consisting of: longifoles, α-selines, β-selines, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β -Bisabolic and β-macrocarpenes. Further preferred embodiments, in particular preferred combinations of VOCs which can be used for the calibration solution, have been described above.

The invention also relates to the use of the calibration solution for calibrating a measuring device for testing maize plants, corn cobs and / or corn kernels for volatile organic substances. In particular, the calibration solution can be used in the method according to the invention.

Examples

1. Materials

The VOCs used for the calibration solution are either commercially available or can be prepared by synthesis or extraction.

For example, selenins (both isomers alpha and / or beta) may be obtained by extracting, for example, celery (Apium graveolens), pepper (Piper nigrum), from Sweet Annie essential oil (Artemisia annua) or Calamus (Acorus calamus). Alpha and Beta Selines can also be obtained from Sellery Oil of Roth (Karlsruhe, Germany). For celery also see: Journal of Food Science and Technology, 200, 37, 6, 631-635; Food Chemistry, 2010, 120, 230-234; Parasitol Res 2008, 104, 107-115. For pepper see: African Journal of Biotechnology 2009, 8, 424-431. Beta-Bisabol can be obtained from Santa Cruz Biotechnology, Inc. USA. Longifolene can be obtained from Sigma-Aldrich, DE, or Metha Oil Industrie (India).

2. Determination of suitable VOCs

Commercial hybrid corn (Ronaldinio, KWS) was grown under greenhouse conditions. The plants were grown on an autoclaved soil-sand mixture. At the time of flowering (BBCH 65), spore suspensions of Fusarium graminearum, F. verticillioides and F. subglutinans as well as mixed suspensions were injected into the scar canal. The control plants were treated with autoclaved water. The dynamic procedure of the VOC collection was carried out over a period of 24 hours at 8-day intervals on the piston. This was an open

Circulation system (English, open loop) established with a continuous air flow. For the static collection of the volatile components by SPME technique, the flasks were harvested after 24 days. Furthermore, the time course of the VOC synthesis was investigated in a time series experiment with harvest times at intervals of 4 days.

The systematic distribution of VOCs within the infected maize plants was investigated by sampling the leaf material.

Analyzes were performed on an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass spectrometer. To identify the components, the mass spectra were compared with commercial and / or standard scientific mass spectrometric databases. A proprietary Perl script for data processing was used, followed by a Kruskal-Wallis rank sum test according to Siegel & Calstellan (1988), supra.

Quoted literature:

DE102009011102 Al;

FR 2650475 AI;

DE 19950396 C2; DE 10002880 Cl;

WO 02/32222 AI;

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Claims

claims

A method of producing maize plants, corn cobs and / or corn kernels with reduced fungal attack, comprising the steps of:

(i) providing maize plants, corn cobs and / or maize kernels, the maize plants having reached at least the growth stage BBCH (Federal Biological Research Center for Agriculture and Forestry, Federal Plant Variety and Chemical Industries) Coding 65, whereby BBCH-65 by a flower of the upper and lower Panicle branches and completely pushed scarring threads is characterized

(ii) testing the corn plants, corn cobs and / or corn kernels of step (i) for at least two volatile organic compounds (VOCs) selected from the group consisting of: longifols, α-selins, β-seleins, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovats index of 1445, β-bisabolites and β-macrocarps, the presence of volatile organic substances indicating fungal infestation,

(iii) (A) treating the corn plants from step (ii) with fungal infestation with spray (s) effective against the fungus found in step (ii); or

(B) separating corn plants, corn cobs and / or corn kernels from step (ii) with fungal infestation from those without fungal infestation, and

2. The method according to claim 1, wherein the fungal attack by Fusarium spp. is caused, in particular by a fungus from the group consisting of: Fusarium graminearum, Fusarium subglutinans, Fusarium verticillioides, and mixtures thereof, preferably Fusarium graminearum and / or Fusarium verticillioides.

3. The method according to claim 1 or 2, wherein at least beta-selenium and beta-macrocarpene is tested.

4. Method according to one of the preceding claims, wherein the instrument for testing the volatile organic substances is selected from the group comprising: gas chromatographs (GC), gas chromatographs coupled with mass spectrometers (GC-MS), ion mobility spectrometers (IMS), Starkfeld Ion mobility spectrometers with asymmetric waveforms (FAIMS), electrochemical sensors, electrochemical sensor arrays, colorimetric sensors, and electric noses.

5. A method according to any one of the preceding claims, wherein the testing is done by aspirating air from the environment (headspace) of the maize plants, corn cobs and / or corn kernels into the meter and testing this air for volatile organics.

6. The method according to any one of the preceding claims, wherein

the corn plants, corn cobs and / or corn kernels are provided in step (i) in batches or in a continuous process, wherein the maize plants, corn cobs and / or corn kernels are tested in step (ii) batchwise or in a continuous process; and

wherein the step in (iii) comprises separating the affected and unaffected corn plants, corn cobs and / or corn kernels in batches in which the respective volatile organic substances are detected / not detected; or the process step in (iii) comprises the continuous separation of fungal infested and non-infested maize plants, corn cobs and / or corn kernels.

7. The method according to any one of the preceding claims, wherein the separation of fungus-affected and unaffected maize plants, corncobs and / or corn kernels by means of an agricultural machine, preferably by means of a harvester takes place.

8. A method of detecting fungal infestation in maize plants, corn cobs and / or corn kernels, comprising testing maize plants, corn cobs and / or maize kernels for at least two volatile organic compounds (VOCs) selected from the group consisting of: longifolene, alpha-selenin, beta Selines, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, ß-bisabolene and ß-macrocarpene, wherein the presence of the volatile organic substances indicates a fungal infection,

where the corn plants have reached at least the growth stage BBCH (Federal Biological Research Center for Agriculture and Forestry, Federal Plant Variety Office and Chemical Industry) Coding 65, whereby BBCH -65 is characterized by a flowering of the upper and lower panicle branches and fully pushed scar tissue; Preferably, the testing of maize plants, corn cobs and / or corn kernels according to step (ii) is as defined in any one of claims 1-5.

9. A system for testing maize plants, corn cobs and / or maize kernels for volatile organic compounds (VOCs), the system comprising a measuring device based on at least two volatile organic substances selected from the group consisting of: longifolene, α-selinin, beta Seleins, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, ß-bisabolene and ß-macrocarpenes, is calibrated.

10. System according to claim 9, wherein

(i) the system is mounted on an agricultural utility vehicle, or on a mobile drone, the system preferably being designed in such a way that sampling takes place in the vicinity of the corncob, or

(ii) the system is installed in a sorting plant, the sorting plant sorting maize plants, corn cobs and / or maize kernels, and the measuring device sends measurement signals to a control element controlling the sorting plant so that the maize plants, maize cob and / or maize kernels of which are separated without fungal attack.

A system according to claim 9 or 10, comprising a calibration unit containing a calibration solution, wherein the calibration solution contains a mixture of VOCs according to the invention and the system can automatically access this calibration solution to automatically perform calibration processes at appropriate intervals.

12. The system according to claim 9, wherein the measuring device is selected from the group consisting of: gas chromatographs (GC), gas chromatographs coupled with mass spectrometer (GC-MS), ion mobility spectrometers (IMS), high field ion mobility Asymmetric waveform (FAIMS) spectrometers, electrochemical sensors, electrochemical sensor arrays, colorimetric sensors, and electrical noses.

13. Use of the system according to any one of claims 9-12 for testing maize plants, corn cobs and / or corn kernels for volatile organic compounds (VOCs) for detecting fungal infestation, wherein the maize plants at least the growth stage BBCH (Federal Biological Research Center for Agriculture and Forestry, Bundessortenamt and Chemical Industry) coding 65, whereby BBCH-65 is characterized by a bloom of the upper and lower panicle branches and completely shifted scar threads.

14. Use of a calibration solution comprising at least two volatile organic compounds (VOCs) from the group consisting of: longifols, α-selines, β-selines, a sesquiterpene with a Kovatsindex of 1442, a sesquiterpene with a Kovatsindex of 1445, β-bisabolites and β-macrocarpenes, for calibrating a measuring device for testing maize plants, corn cobs and / or maize kernels for volatile organic substances for detecting fungal infestation, the maize plants coding for at least the growth stage BBCH (Federal Biological Research Center for Agriculture and Forestry, Federal Office of Plant Varieties and Chemical Industries) 65, whereby BBCH-65 is characterized by a flowering of the upper and lower panicle branches and completely pushed scar threads.