NL2011134C2

NL2011134C2 - A process for determining the presence of a causative agent of black spot on and/or in a plant or a plant part from the genus daucus.

Info

Publication number: NL2011134C2
Application number: NL2011134A
Authority: NL
Inventors: Peter Albert Balk; Nathalie Verhoef
Original assignee: Nsure Holding B V
Priority date: 2013-07-11
Filing date: 2013-07-11
Publication date: 2015-01-13

Description

A process for determining the presence of a causative agent of black spot on and/or in a plant or a plant part from the genus Daucus.

FIELD OF THE INVENTION

The present invention relates to the field of crop sciences, specifically the occurrence of black spot on carrots. More specifically, the present invention relates to a novel method to assess whether carrots will develop black spot disease during or (shortly) after storage. The invention discloses for the first time the possibility to determine the presence of a causative agent of black spot by assessing the expression profile of certain mRNA’s that are native to the plant. In other words, the causative agent of black spot is not detected directly by detection of a nucleic acid, protein or expression profile of the causative agent itself, but by detection of the reaction of the plant on such causative agent.

BACKGROUND OF THE INVENTION

An important problem in organic carrot production, specifically in the Netherlands is the occurrence of black spots during storage. Several different fungal pathogens cause similar symptoms, which are collectively known as black spot diseases, herein often referred to as “black spot”; the terms “black spot”, “black spots” and “black spot diseases” are used interchangeably herein. Several fungi have been identified as being associated with black spot diseases, such as Alternaria radicina, Mycocentrospora acerina, Chalaropsis thielavioides, Chalara elegans, Rhizoctonia destructans and Rhexocercosporidium carotae (Voorrips, et al, 2006).

Carrot is one of the most important crops in organic vegetable production, in terms of both cultivated area and production value. Especially when mature carrot roots are harvested before winter and held in refrigerated storage for several months, returns are profitable. In the last years however, black spots are observed on stored carrots in the Netherlands. When more than 5% of the carrots are affected, the lot is rejected for the fresh market. Specifically in organic farming, the possibilities to control diseases are limited.

Since black spot often develops during storage, or even becomes apparent after (cold) storage, there is a great need for an assay to determine the presence of a causative agent of black spot before or during storage. Such assay would enable to determine whether a charge of carrot would be worthwhile storing, or should e g. be processed instantly.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides a process for determining the presence of a causative agent of black spot on and/or in a plant or a plant part from the genus Daucus, more preferably a Daucus carota, more preferably Daucus carota sativus comprising: - providing a nucleic acid sample of the plant or plant part, - measuring the expression level of a set of at least two mRNA indicator transcripts native to the plant in the nucleic acid sample that are indicative for the presence of a causative agent of black spot, and optionally, - identifying and selecting a plant or a plant part which comprise a predetermined expression level of the set of at least two mRNA indicator transcripts, and - separating a plant or a plant part having an expression profile substantially different from a predetermined reference value from an otherwise identical plant or plant part wherein no causative agent of black spot is present.

According to an embodiment, the nucleic acid sample is provided from a tissue homogenate and the nucleic acid is preferably stabilized on a solid carrier.

According to an embodiment, a nucleic acid sample comprising a pool of nucleic acid samples obtained from at least five, ten, fifteen, twenty, twenty-five, or at least thirty different plants or parts of different plants is measured.

The present invention further provides for the use of set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part for measuring the presence of a causative agent of black spot on and/or in a plant or a plant part.

The present invention further provides a solid carrier comprising a set of at least two nucleic acid molecules attached to the carrier, wherein the nucleic acid molecules are capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part during storage.

The present invention further provides a kit for determining the presence of a causative agent of black spot on and/or in a plant or a plant part, comprising a set of at least two nucleic acid molecules capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part.

According to an embodiment, the kit according to the present invention further comprises at least one of the group consisting of: an instruction for use, a sample buffer, an agent for stabilizing RNA, a control sample, control data, a labeling reagent, a detection reagent, a hybridization agent, an amplification reagent, a primer and/or a probe for detecting housekeeping-gene transcripts, a container, a carrier.

According to an embodiment, the kit according to the present invention further comprises an agent for preparing a nucleic acid sample of a plant or a plant part.

In the embodiments of the present invention, preferably, the at least two mRNA indicator transcripts are mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 5, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 3.

In the embodiments of the present invention, preferably, the set of at least two mRNA indicator transcripts further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 5.

In the embodiments of the present invention, preferably, the causative agent of black spot on and/or in the plant or plant part is at least one fungus, preferably of the group consisting of Chalaropsis, Mycentrospora, Chalara, Alternaria, Rhizoctonia, and Rhexocercosporidiun, more preferably Alternaria radicina, Mycocentrospora acerina, Chalaropsis thielavioides, Chalara elegans, Rhizoctonia destructans and Rhexocercosporidium carotae.

In the embodiments of the present invention, preferably, the plant part is a root, preferably a root vegetable, more preferably a carrot.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a novel method to assess whether carrots will develop black spot disease during of (shortly) after storage. It is believed that this invention for the first time discloses the possibility to determine the presence of a causative agent of black spot by assessing the expression profile of certain mRNA’s that are native to the plant. In other words, the causative agent of black spot is not detected directly by detection of a nucleic acid, protein of expression profile of the causative agent itself, but by detection of the reaction of the plant on such causative agent. Surprisingly, by analysis of the reaction of the plant on a causative agent of black spot, it is possible to assess whether black spot may develop during or (shortly) after storage.

Accordingly, in a first aspect the present invention provides a process for determining the presence of a causative agent of black spot on and/or in a plant or a plant part from the genus Daucus, more preferably a Daucus carota, more preferably Daucus carota sativus comprising: - providing a nucleic acid sample of the plant or plant part, - measuring the expression level of a set of at least two mRNA indicator transcripts native to the plant in the nucleic acid sample that are indicative for the presence of a causative agent of black spot, and optionally, - identifying and selecting a plant or a plant part which comprise a predetermined expression level of the set of at least two mRNA indicator transcripts, and - separating a plant or a plant part having an expression profile substantially different from a predetermined reference value from an otherwise identical plant or plant part wherein no causative agent of black spot is present.

The nucleic acid sample in the embodiments of the present invention may be any kind of nucleic acid sample comprising at least a nucleic acid. The nucleic acid may be any known to the person skilled in the art, such as but not limited to DNA, cDNA, RNA, mRNA. Preferably, the nucleic acid sample comprises mRNA. The nucleic acid sample may be purified to some extent. Purification is only necessary to facilitate the measurement of the expression level of an mRNA indicator transcript. The person skilled in the art knows methods available for the purification of a nucleic acid. Purification may not be necessary; it may suffice to disrupt the natural environment of the nucleic acid before or during the method according to the invention. Purification may be performed by any means known to the person skilled in the art, such as the isolation of poly-[A]-RNA, or the isolation of total RNA. The nucleic acid sample may be obtained from fresh tissue, or may be obtained from a tissue sample that has been preserved. Preservation may be done by any method known by the person skilled in the art that will preserve the integrity of the nucleic acid such that the expression level of an mRNA indicator transcript can be measured, such as preservation in lowered temperatures such as in a freezer at -50, -80, -100, -130 or -150 degrees Celsius or in liquid nitrogen at -196 degrees Celsius. A preferred nucleic acid sample is prepared from a tissue homogenate. The tissue homogenate may be prepared by any means known to the person skilled in the art; preferably the agent to prepare the tissue homogenate also preserves the nucleic acid, preferably the mRNA. This allows storage of the tissue homogenate without degradation of the nucleic acid. Preferably, the nucleic acid sample is stabilized on a solid carrier; preferably a tissue homogenate is made and is spotted on a solid carrier, preferably a filter paper card that protects from nucleases, such as an FT A® card (Whatman FTA® technology). Such filter paper card captures the nucleic acid present in the sample, preferably the tissue homogenate; agents that preserve the nucleic acid are also present in the filter paper. The sample is preferably allowed to dry after spotting on the filter paper, preferably at least one hour. After drying, the solid carrier can be stored, preferably desiccated, until further processing steps according to invention.

The nucleic acid sample may be provided from several individual plants or plant parts of several individual plants, or more preferable from a batch of plants or plant parts. Accordingly, a nucleic acid sample preferably comprising a pool of nucleic acid samples obtained from at least two, more preferably, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, twenty-five, or at least thirty different plant or plant parts, preferably from a batch, is measured in the method according to the invention. The plant tissue from which the sample is taken may be the entire plant, or may be any plant, such as a skin sample from a root such as a carrot. The plant tissue from which the sample is taken may be pooled before the nucleic acid sample is obtained, or the nucleic acid samples may be obtained and pooled after obtaining the nucleic acid sample.

Sampling (taking of a tissue sample for preparing a nucleic acid sample) may be performed at any time shortly before and during storage, sampling may be performed once, or multiple times. Preferably, sampling is performed shortly after harvest; preferably at approximately one day, one week, two weeks, three weeks and most preferably four weeks after harvest. Sampling may also be performed once, or multiple times during storage or may be performed during cultivation, after harvest and/or during storage.

In all embodiments of the present invention, at least two mRNA indicator transcripts are native to the plant. Preferably, at least three, four, five, six, seven, eight, nine or ten mRNA indicator transcripts are native to the plant; more preferably all mRNA indicator transcripts are native to the plant.

In all embodiments of the present invention, the expression level of the at least two indicator mRNA transcripts may be determined using any means known to the person skilled in the art. Such means are well-known in the art and include, but are not limited to, quantitative PCR methods, preferably quantitative RT-PCR, or nucleic acid hybridization based methods (for example microarray hybridization). Quantitative PCR (qPCR) may be carried out by conventional techniques and equipment, well known to the person skilled in the art, optionally in multiplex reactions and/or in quantitative competitive reactions. Preferably, labeled primers or labeled oligonucleotides or intercalating dyes such as SYBR Green are used to quantify the amount of reaction product. Other techniques capable of quantifying relative and absolute amounts of mRNA in a sample, such as NASBA (Nucleic Acid Sequence Based Amplification), may also be suitably applied. Another convenient system for quantification is the immunolabeling of the primers, followed by an immuno-lateral flow system (NALFIA) on a pre-made strip. As a positive control for the RNA isolation, reverse transcriptase reaction, amplification reaction and detection step, amplification and detection of a constitutively expressed housekeeping gene is preferably included in the assay, such as ribosomal (e.g. 18S or 25S) rRNA’s, actin mRNA, tubulin mRNA or GAPDH mRNA; preferably one or more of alpha tubuline (SEQ ID NO: 8), NADH (SEQ ID NO: 9) and/or ubiquitin (SEQ ID NO: 10) are used for such normalization. Primers may e.g. be labeled with direct labels such as FITC (fluorescein), Texas Red, Rhodamine and others or with tags such as biotin, lexA or digoxigenin which may be visualized by a secondary reaction with a labeled streptavidin molecule (for instance with carbon or a fluorescent label) or a labeled antibody (labeled with fluorescent molecules, enzymes, carbon, heavy metals, radioactive isotopes or with any other label).

Nucleic acid hybridization based methods include comparative hybridization performed on mRNA or cDNA populations obtained from a plant or sample thereof, using a set of indicator gene sequences (probes), which may optionally be tagged or labeled for detection purposes, or may be attached to a solid carrier such as a DNA array or microarray. Suitable methods for microarray detection and quantification are well described in the art. To construct a DNA microarray, nucleic acid molecules (e.g. single stranded oligonucleotides according to the invention) are attached to a solid support at known locations or “addresses”. The arrayed nucleic acid molecules are complementary to the indicator nucleotide sequences according to the invention, and the location of each nucleic acid on the chip is known. Such DNA chips or microarrays, are well-known in the art. Such arrays may, for example, be produced using mechanical synthesis methods or light-directed synthesis methods that incorporate a combination of photolithographic methods and solid phase synthesis methods. Also methods for generating labeled polynucleotides and for hybridizing them to DNA microarrays are well known in the art.

The expression level of indicator mRNA transcripts may be depicted in any way known the person skilled in the art. The expression level may e.g. be depicted in absolute numbers of mRNA molecules in a specific amount of sample; or e.g. as a percentage of the mRNA The expression level may also be depicted in a relative way compared to a reference sample, such as a percentage or ratio in view of said reference sample.

Identifying and selecting a plant or a plant part which comprises a predetermined expression level of the set of at least two mRNA indicator transcripts may be performed according to any method known to the person skilled in the art. One may e.g. consider for positive identification and selection, that the predetermined expression level of a first mRNA indicator transcript in a subject nucleic acid sample should be at least about two-fold higher than the expression level of the same mRNA indicator transcript in a reference nucleic acid sample (the reference value for the first mRNA indicator transcript) and that the predetermined expression level of a second mRNA indicator transcript in the subject nucleic acid sample should be at least about two-fold lower than the expression level of the same mRNA indicator transcript in a reference nucleic acid sample (the reference value for the second mRNA indicator transcript).

In an embodiment, one may consider for positive identification and selection, that the predetermined expression level of at least half of the mRNA indicator transcripts in a subject nucleic acid sample should be at least about two-fold higher than the expression level of the same mRNA indicator transcripts in a reference nucleic acid sample. A reference nucleic acid sample is preferably from an otherwise identical plant or plant part wherein no causative agent of black spot is present, i.e. a plant or plant part know not to develop black spot. The person skilled in the art may use software tools that are often provided with quantitative PCR kits and/or apparatus to perform the calculations to identify and select a plant or a plant part which comprises a predetermined expression level of a set of at least two mRNA indicator transcripts.

In an embodiment, identifying and selecting a plant or a plant part which comprises a predetermined expression level of the set of at least two mRNA indicator transcripts may be performed by using the ratios of the expression levels of several mRNA indicator transcripts within a single nucleic acid sample or pool of nucleic acid samples for positive identification and selection.

When determining the ratios of the expression levels of several mRNA indicator transcripts within a single nucleic acid sample or pool of nucleic acid samples, preferably the expression level of at least one housekeeping gene, such as alpha tubuline (SEQ ID NO: 8), NADH (SEQ ID NO: 9)and/or ubiquitin (SEQ ID NO: 10) is used for normalization; more preferably two or three of alpha tubuline (SEQ ID NO: 8), NADH (SEQ ID NO: 9) and/or ubiquitin (SEQ ID NO: 10) are used for normalization.

In order to separate a plant or a plant part, one may consider that the expression profile should be substantially different from a predetermined reference value from an otherwise identical plant or pant part wherein no causative agent of black spot is present. One may e.g. consider that for separating, the expression profile should meet the following requirements: the predetermined expression level of a first mRNA indicator transcript in a subject nucleic acid sample should be at least three-fold higher than the expression level of the same mRNA indicator transcript in a reference nucleic acid sample and that the predetermined expression level of a second mRNA indicator transcript in the subject nucleic acid sample should be at least three-fold lower than the expression level of the same mRNA indicator transcript in the reference nucleic acid sample. One may also consider that expression profile, depicted as the ratios of the expression levels of several mRNA indicator transcripts within a single nucleic acid sample or pool of nucleic acid samples should be substantially identical to the expression profile of an otherwise identical plant or pant part wherein no causative agent of black spot is present or wherein one or more causative agent of black spot is present. When determining the ratios of the expression levels of several mRNA indicator transcripts within a single nucleic acid sample or pool of nucleic acid samples, preferably the expression level of at least one housekeeping gene, such as alpha tubuline (SEQ ID NO: 8), NADH (SEQ ID NO: 9) and/or ubiquitin (SEQ ID NO: 10) is used for normalization; more preferably two or three of alpha tubuline (SEQ ID NO: 8), NADH (SEQ ID NO: 9)and/or ubiquitin (SEQ ID NO: 10) are used for normalization.

Preferably, in all embodiments of the present invention, the at least two mRNA indicator transcripts are mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 5, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 3.

Preferably, in all embodiments of the present invention, the set of at least two mRNA indicator transcripts further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 5.

Other preferred sets of mRNA indicator transcripts according to the present invention for all embodiments according the invention are listed here below. A preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2 and SEQ ID NO: 3.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2 and further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 4 -SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 and further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 4 -SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3 and further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 4 -SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

Another preferred set of mRNA indicator transcripts according to the present invention comprises the mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 and further comprises at least one, two, three, four, five, six or at least seven mRNA indicator transcripts that each comprise or consist of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 4 -SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 5.

In all embodiments of the present invention, a gene whose expression is preferably used for determining the relative expression level (normalization) and/or ratio of expression of an mRNA indicator transcript according to the invention is at least one housekeeping gene; preferably alpha tubuline (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 8), NADH (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 9) and/or ubiquitin (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 10) are used; more preferably two or three of alpha tubuline (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 8), NADH (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 9) and/or ubiquitin (a nucleic acid molecule that comprises or consists of a nucleic acid molecule with a sequence identity of at least 70% with SEQ ID NO: 10) are used.

In all embodiments of the present invention, the sequence identity is preferably at least 70%, more preferably at least 75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% and most preferably 100%.

Sequence identity is preferably defined as known in the art, as a relationship between two or more nucleic acid sequences by comparing the sequences. In the art, the percentage of "identity" indicates the degree of sequence relatedness between nucleic acid residues as determined by the match between strings of such sequences. Preferably, the percentage of identity is determined by comparing the whole SEQ ID NO as identified herein. However, part of such sequence may also be used. In this context, “part” means at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of its length. Preferably 100% is used. "Sequence identity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity are codified in publicly available computer programs. Preferred computer program methods to determine identity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1):387 (1984)), BestFit and FASTA (Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990). The BLAST 2.0 family of programs which can be used for database similarity searches includes: BLASTP for protein query sequences against protein database sequences. The well-known Smith Waterman algorithm may also be used to determine identity.

Another preferred method to determine sequence identity is by using the algorithm Needleman-Wunsch (Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453, Kruskal, J. B. (1983) An overview of sequence comparison In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley). The following website could be used: http://www.ebi.ac.uk/Tools/emboss/align/index.html.

Definitions of the parameters used in this algorithm are found at the following website:: http://emboss.sourceforge.net/docs/themes/AlignFormats.html#id.Preferably, using this algorithm, Gappenalty is 10.0 and Extend_penalty is 0.5.

Preferably, the causative agent of black spot on and/or in the plant or plant part is at least one fungus, preferably of the group consisting of Chalaropsis, Chalara, Mycentrospora, Alternaria, Rhizoctonia, and Rhexocercosporidiun, more preferably Alternaria radicina, Mycocentrospora acerina, Chalaropsis thielavioides, Chalara elegans, Rhizoctonia destructans and Rhexocercosporidium carotae..

Preferably, the plant part is a root, preferably a root vegetable, more preferably a carrot.

In a second aspect, the present invention provides for the use of set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part for measuring the presence of a causative agent of black spot on and/or in a plant or a plant part.

The set of at least two mRNA indicator transcripts, the causative agent, the plant or plant part and the method for measuring the presence of a causative agent are preferably those defined in the first aspect of the present invention.

In a third aspect, the present invention provides for a solid carrier comprising a set of at least two nucleic acid molecules attached to the carrier, wherein the nucleic acid molecules are capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part during storage.

The carrier may be any carrier known to the person skilled in the art that is capable of attaching a nucleic acid molecule. A carrier may for example be an array or microarray or a DNA chip, comprising nucleotides on a glass, plastic, nitrocellulose or nylon sheet, silicon or any other solid surface, all are well known in the art and for instance described in Bowtell and Sambrook, 2003 and in Ausubel et al., 2004. A carrier according to the current invention comprises at least two, such as at least 2, 3, 4, 5, 10, 15, 20, 25, 30, or more (oligo-)nucleotide probes capable of selectively hybridizing with indicator genes (mRNA or cDNA) present in a sample. In addition, the carrier may comprise or more (oligo-)nucleotide probes capable of selectively hybridizing with mRNA reference molecules such as transcripts of constitutively expressed housekeeping genes, such as ribosomal (e.g. 18S or 25S) rRNA’s, actin mRNA, tubulin mRNA or GAPDH mRNA.

Hybridization of nucleic acid molecules is known to the person skilled in the art. The following conditions are given as an example, the person skilled in the art knows how and can modify the conditions. Low to very high stringency conditions can be prehybridization and hybridization at 42 degrees Celsius in 5 x SSPE, 0.3% SDS, 200 microgram/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard detection procedures.

The carrier material may finally be washed three times each for 15 minutes using 2 x SSC, 0.2% SDS at 45 degrees Celsius for very low stringency, at 50 degrees Celsius for low stringency, at 55 degrees Celsius for medium stringency, at 60 degrees Celsius for medium-high stringency, at 65 degrees Celsius for high stringency, and at 70 degrees Celsius for very high stringency.

In a fourth aspect, the present invention provides for a kit for determining the presence of a causative agent of black spot on and/or in a plant or a plant part, comprising a set of at least two nucleic acid molecules capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part; preferably, the at least two nucleic acid molecules capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part are PCR primers.

The set of at least two mRNA indicator transcripts, the causative agent, the plan or plant part and the method for measuring the presence of a causative agent are preferably those defined in the first aspect of the present invention.

The at least two nucleic acid molecules capable of hybridizing to a set of at least two mRNA indicator transcripts that are indicative for the presence of a causative agent of black spot on and/or in a plant or a plant part are preferably those defined in the third aspect of the present invention.

The kit according to the invention may further comprise at least one of the group consisting of: an instruction for use, a sample buffer, an agent for stabilizing RNA, a control sample, control data, a labeling reagent, a detection reagent, a detection agent for use in PCR, such as SYBR Green, a hybridization agent, an amplification reagent, a primer and/or a probe for detecting housekeeping-gene transcripts, a container, a carrier.

The kit according to the invention may further comprise an agent for preparing a nucleic acid sample of a plant or a plant part.

Optionally, the kit may include tissue samples or nucleic acid samples suitable as reference samples. A kit may comprise a carrier to receive therein one or more containers, such as tubes or vials. The kit may further comprise unlabeled or labelled oligonucleotide sequences according to the invention, e.g. to be used as primers, probes, which may be contained in one or more of the containers, or may be present on a carrier, preferably a carrier as defined herein. The oligonucleotides may be present in lyophilized form, or in an appropriate buffer. One or more enzymes or reagents for use in isolation of nucleic acids, purification, restriction, ligation and/or amplification reactions may be contained in one or more of the containers. The enzymes or reagents may be present alone or in a mixture, and in lyophilised form or in appropriate buffers.

The kit may also contain any other component necessary for carrying out the present invention, such as enzymes (such as preferably reverse transcriptase and a thermostable polymerase), pipettes, plates, nucleic acids (preferably labelled probes), nucleoside triphosphates, filter paper, gel materials, transfer materials, electrophoresis materials, visualization materials (preferably dyes, labelled antibodies or -enzymes) and/or autoradiography supplies.

GENERAL DEFINITIONS

The term “gene” is preferably defined herein as a DNA fragment comprising a region that can be transcribed into an RNA molecule (e.g. an mRNA molecule) in a suitable in vivo or in vitro system such as microbiological cell, said region being operably linked to a suitable regulatory region, such as a promoter. A gene may comprise additional regulatory sequences such as a 5’-leader sequence, a signal sequence, a transcription regulatory region, a polyadenylation region and/or a termination site. Expression of a gene refers to the process wherein a polynucleotide, preferably a DNA, is transcribed into an RNA molecule depicted as the “transcript”.

An “indicator gene” is preferably defined herein as a gene whose expression level is indicative of the presence of a specific trait; preferably said trait is the presence of a causative agent of black spot.

An “mRNA indicator transcript” is preferably defined herein as an mRNA whose expression level is indicative of the presence of a specific trait; preferably said trait is the presence of a causative agent of black spot.

The term “nucleic acid sample” is preferably defined herein as an amount of nucleic acid, preferably DNA and/or RNA; the sample, may be purified to some extent or the natural environment of the nucleic acid, such as a plant cell, may be disrupted.

The term “plant” refers to any organism of which at least some of the cells comprise chloroplasts. A plant part herein may be any part of the plant, such as but not limited to: a cell, a tissue, an organ, a fruit, a root, a leave, a flower, a flower bud and an anther. A plant cell includes a protoplast, a gamete, a spore, a microspore and a pollen grain.

The term “upregulation” is used interchangeably herein with the tern “induction”; it refers the amount of gene expression and is preferably defined herein as an amount of mRNA transcript that is substantially higher than a predetermined reference value.

The term “downregulation” is used interchangeably herein with the term “reduction” and is preferably defined herein as an amount of mRNA transcript that is substantially lower than a predetermined reference value.

The term “expression level” is herein preferably defined as the amount of mRNA transcript. The term “amount of mRNA transcript” and methods how to measure it are defined later herein.

The term ”a predetermined expression level” is preferably defined herein as a statistically relevant difference, such as at least about a twofold difference compared to a reference value, the difference being relative to e.g. another mRNA indicator transcript according to the invention within the same sample or to the same mRNA indicator transcript in another sample, preferably a said other sample is a sample from a plant or plant part that is known no to develop black spot, preferably at least about a three-fold difference, more preferable a four-fold, five-fold, ten-fold, twenty-fold, thirty-fold, forty-fold, fifty-fold, sixty-fold, seventy-fold, eighty-fold, ninety-fold, hundred-fold, three-log, four-log difference. When comparing expression levels of mRNA indicator genes, the expression levels are preferably normalized using the expression level of a housekeeping gene, preferably a housekeeping gene as described earlier herein, as determined from within the same nucleic acid sample.

The term “reference value” is used herein with respect to the expression level of a subject gene in a subject nucleic acid sample from a plant, relative to a reference nucleic acid sample and is preferably defined herein as the value of the expression level of the identical gene in the reference nucleic acid sample from an otherwise identical plant wherein no causative agent of black spot is present, i.e. a plant or plant part that is known not to develop black spot. When used in relation to an expression profile, the term reference value is construed herein as plural; i.e. the set of reference values of expression levels of several genes in the reference nucleic acid sample. The reference value may also be referred to as the expression level of a housekeeping gene, preferably a housekeeping gene as described earlier herein, as determined from within the same nucleic acid sample. In this case, the expression level of the subject gene is thus measured relative to the expression level of the housekeeping gene within the same sample.

The reference value may also be referred to as the expression level of another mRNA indicator transcript according to the invention within the same sample. In this case comparison is made between the expression levels or ratios of expression levels of two or more different indicator genes to determine an expression profile.

The term “about” when used in association with a numerical value (e.g. about 10) is preferably defined herein as that the value may be the given value (e.g. of 10) plus of minus 10% of the given value.

The term “batch” is preferably defined herein as a collection of harvest plant of plant parts that have been cultivated together, on the same field and have been harvested at the same time.

The term "nucleic acid" is used interchangeable with the term “nucleic acid molecule” and includes RNA, DNA, and cDNA molecules. The term is also used interchangeably with polynucleotide. An oligonucleotide is a short chain nucleic acid molecule. A primer is an oligonucleotide, whether occurring naturally as in a purified restriction digest or produced synthetically, which is capable of acting as a point of initiation of synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is induced, (i.e. in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH). The primer is preferably single stranded for maximum efficiency in amplification.

The term “PCR primer” includes both a degenerate primer and a non-degenerate primer (i.e. of identical nucleic acid sequence as the target sequence to which it hybridizes).

The term “Oligonucleotide” refers to a nucleic acid fragment suitable for use as PCR primer or hybridization probe, e.g. coupled to a carrier in a nucleic acid microarray.

The term “DNA Microarray” or “DNA chip” refers to a series of known DNA sequences (oligonucleotides or oligonucleotide probes) attached in a regular pattern on a solid surface, such as a glass slide, and to which a composition consisting of or comprising target sequences are hybridized for identification and/or quantification.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". The word “about” or “approximately” when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. FIGURE LEGENDS Figure 1.

Figure 1A depicts the expression profile of four selected mRNA indicator transcripts for three different batches of carrots (batches 30, 52 and 6).

Figure IB depicts the quality, related to the presence of black spot after storage.

SEQUENCES

Table 1. Sequences as set forth in the Sequence Listing

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry. Such techniques are described in Sambrook etal. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel etal. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; Ausubel et al., Current protocols in Molecular Biology, Wiley Interscience, 2004; and in Volumes I and Π of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.).

EXAMPLES

Selection of genes indicative for the presence of a causative agent of black spot.

To identify carrot specific genes that can be used to predict black spot during cold storage by determining the presence of a causative agent of black spot, skin tissue samples, each from 25 carrots from a total of 50 batches of carrots of the variety Nerac were taken at several time points around harvest en during storage directly after harvest in 2010 and were frozen at -196 degrees Celsius. The batches of carrots were stored at a diversity of commercial cooling facilities degrees. In 2011 and 2012, the assay was performed and validated and carrot batches were visually scored for the presence of black spots.

Total RNA was isolated using the method of Chang et al., 1993 from frozen skin tissue samples of known poor-storable batches and known well-storable batches. Subsequently, the transcriptome of the samples was sequenced. By comparing the transcriptomes (using CLC Bio software) of good storable batches with the transcriptomes of poor storable batches, genes were identified whose expression was modulated at time of sampling in batches that later developed black spot during storage; the sequences of several genes are depicted in Table 1 and in the sequence listing. The CLC Bio software extracts from the raw transcriptome data which RNA molecules are present in the sample and in which quantity. By comparing samples from known poor-storable batches and known well-storable batches, the correlation between the degree of affection (black spot) and the corresponding expression profile was made.

Measurement of the presence of a causative agent of black spot using selected mRNA indicator transcripts. cDNA was synthesized from frozen RNA samples, isolated at harvest time, of several batches (using iScript, Bio-Rad) and expression levels of the selected indicators was measured using real-time RT-PCR (CFX real-time PCR detection system, Bio-Rad) using standard protocols (iQ SYBR Green Supermix, Bio-Rad). Using this technique, indicators have also been validated on batches harvested in subsequent years (2011, 2012). Finally, an index based on the relative expression levels of the indicators and related to the risk for development of black spots was developed to assign each batch to a risk stage; high (black), medium (white) or low (low).

Figure 1A depicts the expression profile of four selected mRNA indicator transcripts for three different batches of carrots (batches 30, 52 and 6). The expression profile was determined on frozen skin tissue samples using the procedure described here above. The risk stage is depicted as stated here above; high (black), medium (white) or low (low). Based on the expression profile batch, 30 was assigned to be of good quality, batch 52 of intermediate quality and batch 6 as a batch with a high risk for development of black spots during storage.

Figure IB depicts the quality, related to the presence of black spot after storage. After storage, batch 30 had not developed black spot and was depicted a a good quality batch; batch 52 had developed some black spots and was depicted as a intermediate quality batch; batch 6 developed a lot of black spots and was depicted as a batch with high risk for developing black spot during storage.

Indicators Pel, Pe2, Pe4 en Pe5 correspond to mRNA indicators SEQ ID NO: 4, 6, 7 and 5, respectively.

REFERENCE LIST 1. Voorrips, et al, Black spot diseases in carrot. Proceedings of the European Joint Organic Congress, Odense, Denmark, 30-31 May 2006 2. Chang, S., Puryear, J and Cairney J. (1993) A simple and efficient method for isolating RNA from pine trees. Plant. Mol. Biol. Rep. 11:113-116 3. Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993 4. Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994 5. Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987 6. Sequence Analysis Primer Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H, and Lipman, D., SIAM J. Applied Math., 48:1073, 1988 7. Devereux, J., et al., Nucleic Acids Research 12 (1):387, 1984 8. Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990 9. Needleman, S. B. and Wunsch, C. D., 1970; J. Mol. Biol. 48, 443-453 10. Kruskal, J. B., 1983; An overview of sequence comparison. In D. Sankoff and J. B. Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley 11. Sambrook et al., 1989; Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press

12. Sambrook and Russell, 2001; Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY 13. Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual, Cold Spring Harbor Laboratory Press, 2003 14. Ausubel et al., Current Protocols in Molecular Biology, Current Protocols, USA, 1994 15. Ausubel et al., Current protocols in Molecular Biology, Wiley Interscience, 2004. 15. Brown, 1998; Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); Nucleic Acid Hybridization (Hames and Higgins, eds.)

Claims

A method for determining the presence of a black spot agent on and / or in a plant or plant part of the genus Daucus, preferably a Daucus carota, more preferably Daucus carota sativus comprising: - providing a nucleic acid sample of the plant or part of the plant, - measuring the expression level of a set of at least two plant-specific mRNA indicator transcripts in the nucleic acid sample which are indicative of the presence of a black spot agent, and optionally - identifying and selecting a plant or a plant part containing a predetermined expression level of the set of at least two mRNA indicator transcripts, and - separating a plant or a plant part with an expression profile substantially different from a predetermined reference value of an otherwise identical plant or plant part in which no black spot causative agent is present.

The method of claim 1, wherein the nucleic acid sample is stretched from a tissue homogenate and the nucleic acid is preferably stabilized on a solid support.

The method of claim 1 or 2, wherein a nucleic acid sample comprising a pool of nucleic acid samples obtained from at least five, ten, fifteen, twenty, twenty-five or at least thirty different plants or different plants is measured.

Use of a set of at least two mRNA indicator transcripts indicative of the presence of a black spot agent on and / or in a plant or plant part for measuring the presence of a black spot agent on and / or a plant or part of a plant.

A kit for determining the presence of a black spot agent on and / or in a plant or plant part, comprising a set of at least two nucleic acid molecules that hybridize to a set of at least two mRNA indicator transcripts indicative of the presence of a causative agent of black spot on and / or in a plant or part of a plant.

The kit of claim 5, further comprising at least one of the group consisting of: an instruction for use, a sample buffer, an RNA stabilizer, a control sample, control data, a labeling reagent, a reagent for detection, a detection agent for use in PCR, such as SYBR Green, a hybridization agent, an amplification reagent, a primer and / or a probe for detecting housekeeping gene transcripts, a container, a carrier.

The kit of claim 5 or 6, further comprising an agent for preparing a nucleic acid sample from a plant or plant part.

A method according to claims 1-3, the use according to claim 4 and the kit according to claims 5-7, wherein the at least two mRNA indicator transcripts are mRNA indicator transcripts each comprising or consisting of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 28, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 10, more from the group consisting of SEQ ID NO: 1 : 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 5, more preferably from the group consisting of SEQ ID NO: 1 SEQ ID NO: 3.

The method of claims 1-3 and 8, the use of claims 4 and 8 and the kit of claims 5-8, wherein the set of at least two mRNA indicator transcripts further comprises at least one, two, three, four, five, six, seven or at least eight mRNA indicator transcripts each comprising or consisting of a nucleic acid molecule with a sequence identity of at least 70% with a sequence selected from the group consisting of SEQ ID NO: 4 - SEQ ID NO: 28, more preferably the group consisting of from SEQ ID NO: 1 - SEQ ID NO: 10, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 7, more preferably from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 1 : 5.

Method according to claims 1-3 and 8-9, the use according to claims 4 and 8-9 and the kit according to claims 5-9, wherein the black spot agent on and / or in the plant or plant part is at least one fungus , preferably from the group consisting of Chalar opsis, Chalara, Mycentrospora, Alt ernoria, Rhizoctonia and Rhexocercosporidin.

Method according to claims 1-3 and 8-10, the use according to claims 4 and 8-10, the carrier according to claims 5 and 8-10, and the kit according to claims 5-10, wherein the plant part is a root, preferably is a root vegetable, more preferably a carrot.