# Patent

## Title
Dimeric diagnostic arrays

## Abstract

The invention provides dimeric diagnostic arrays and methods for their use.

## Background
RELATED APPLICATION(S)
This application claims the benefit of priority of U.S. Provisional Application Ser. No. 61/476,143 filed on Apr. 15, 2011, which application is herein incorporated by reference.


Sequence Listing
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Sep. 4, 2012, is named 08035006.txt and is 68,827 bytes in size.
BACKGROUND
Detection and identification of pathogens, e.g., microbial plant pathogens, poses a challenge because different pathogens may infect the same host concurrently and may produce similar symptoms. In the absence of clear distinctive symptoms and signs, plant disease diagnosticians may use the host identity, time of the year and prevailing weather conditions to associate the pathogen with the disease. Accurate pathogen identification is the first step in disease management. Misidentification of a pathogen may lead to poor disease control, crop damage and ultimately reduced yield. There is therefore a need for improved disease surveillance, more rapid diagnoses, and accurate remedial measures in the shortest time possible.
SUMMARY OF CERTAIN EMBODIMENTS OF THE INVENTION
Certain embodiments of the present invention provide an array that comprises a first plurality of dimeric probes that hybridize to a first target nucleic acid sequence, wherein the dimeric probes each comprise a first hybridizing nucleic acid sequence and a second hybridizing nucleic acid sequence linked together, wherein the first and second hybridizing nucleic acid sequences are the same and hybridize to the first target nucleic acid sequence. In certain embodiments, the dimeric probe comprises a third hybridizing nucleic acid sequence that hybridizes to the first target nucleic acid sequence and is the same as the first a second hybridizing nucleic acid sequences.
Redundancy in the array may improve accuracy and analytical power through over-representation. As used herein, the phrase a “plurality of dimeric probes” means at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) probes that hybridize to the same target nucleic acid sequence.
In certain embodiments, the first plurality of dimeric probes comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) probes.
In certain embodiments, the first and second hybridizing nucleic acid sequences are linked directly together.
In certain embodiments, the first and second hybridizing nucleic acid sequences are linked together via a nucleic acid linker sequence.
In certain embodiments, the nucleic acid linker sequence is a poly-adenine linker (e.g., about 5-15, e.g., about 10 nucleotides in length).
In certain embodiments, the dimeric probes are about 40-60 nucleotide in length.
In certain embodiments, the dimeric probes are about 40-48 nucleotide in length.
In certain embodiments, the dimeric probes are about 50-58 nucleotide in length.
In certain embodiments, the array comprises a second plurality of dimeric probes that hybridize to a second target nucleic acid sequence.
In certain embodiments, the second plurality of dimeric probes comprises at least two (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) probes.
As redundancy may improve the accuracy and analytical power of the array, probes that recognize different target nucleic acid sequences in the same pathogen may be used.
Accordingly, in certain embodiments, the first and second pluralities of probes hybridize to target nucleic acid sequences in the same pathogen.
The array may also be designed to detect more than one type of pathogen. DNA sequences available in publicly accessible databases (e.g., GenBank) allow for the creation of signature probes specific to a species or infra-species target. Accordingly, one skilled in the art may design a dimeric probe as described herein, which is specific to any given species or sub-species, wherein the genomic sequence of the species or sub-species is known (e.g., full or partial), using techniques known in the art or described herein (e.g., Example 1 or 2).
Accordingly, in certain embodiments, the first and second pluralities of probes hybridize to target nucleic acid sequences in different pathogens.
In certain embodiments, the array comprises more than two pluralities of dimeric probes (e.g., 3-6) that hybridize to different target nucleic acid sequences.
In certain embodiments, each plurality of dimeric probes comprises at least two (e.g., 3, 4, 5, 6, 7, 8, 9, 10) probes.
In certain embodiments, the pluralities of probes hybridize to target nucleic acid sequences in the same pathogen.
In certain embodiments, the pluralities of probes hybridize to target nucleic acid sequences in different pathogens.
In certain embodiments, the array comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about two (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33 or 34) different pathogens (e.g., each plurality of dimeric probes hybridizes to a specific target sequence and each target sequence is particular to a given pathogen). In certain embodiments, the array comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about 35 different pathogens. In certain embodiments, the array comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about 50 different pathogens. In certain embodiments, the array comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about 75 different pathogens. In certain embodiments, the array comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about 100 (e.g., about 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1,000, 2,000, 3,000, 4,000, 5,000, 10,000, 25,000, 50,000, 75,000, 100,000, 500,000, etc.) different pathogens.
In certain, embodiments, the target nucleic acid sequence is located in an internal transcribed spacer sequence of an rRNA gene. In certain, embodiments, the target nucleic acid sequence is located in EF1-alpha, Beta-tubulin, RPB1, SSU, or LSU.
In certain embodiments, the pluralities of dimeric probes are selected from the sequences listed in Table 2 or Table 7.
In certain embodiments, the pluralities of dimeric probes are selected from SEQ ID NO:4 to SEQ ID NO:9, SEQ ID NO:19 to SEQ ID NO:24, SEQ ID NO:28 to SEQ ID NO:33, SEQ ID NO:42 to SEQ ID NO:45, SEQ ID NO:48 to SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:73 to SEQ ID NO:301.
In certain embodiments, the pluralities of dimeric probes are selected from SEQ ID NO:4 to SEQ ID NO:6, SEQ ID NO:19 to SEQ ID NO:21, SEQ ID NO:28 to SEQ ID NO:30, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:57, and SEQ ID NO:73 to SEQ ID NO:301.
In certain embodiments, a target sequence is from a fungal, viral, or bacterial pathogen.
In certain embodiments, a target sequence is from a pathogen of turfgrass.
In certain embodiments, a target sequence is from Brumeria graminis, Bipolaris zeicola, Colletotrichum cereal, Eudarluca caricis, Puccinia coronate, Puccinia persistens var triticina, Puccinia striiformis, Puccinia graminis, Puccinia graminis f. sp. Tritici, Pythium volutum, Pythium torulosum, Pythium arrhenomanes, Pythium deliense, Pythium rostratifingens, Pythium rostratum, Pythium aphanidermatum, Pythium myriotylum, Pythium arrhenomanes, Rhizoctonia solani, Ceratobasidium cereal, Waitea circinata, Rhizoctonia zeae, Waitea circinata var. circinata, Rhizoctonia oryzae, Sclerotinia homoeocarpa, Typhula incarnate, Typhula ishikariensis, Gaeumannomyces graminis, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe poae, Gaeumannomyces incrustans, Magnaporthe rhizophila, Magnaporthe salvinii, Microdochium bolleyi, Microdochium nivale, Gleocercospora sorghi, Laetisaria fuciformis, Leptosphaeria korrae, Ophiosphaerella herpotricha, Ophiosphaerella agrostis, Limonomyces roseipellis, Acidovorax avenae, Xanthomonas translucens pv. Poae, Curvularia trifolii, Trichoderma virens, Urocystis agropyri, Ustilago striiformis, Lycoperdon spp, Bovista, Agaricus, Marasmius, Lepiota, Athelia rolfsii, Gibberella zeae, Fusarium solani, Fusarium oxysporum (F. oxysporum), Fusarium spp, Poa annua (RBCL), Lolium perenne (RBCL), Agrostis stolonifera (RBCL), Poa annua (matK), Poa pratensis (matK), Agrostis stolonifera (matK), Motierella elongata, Fusarium equisetti or Waitea circinatavar. zeae. 
In certain embodiments, a target sequence is from Rhizoctonia solani, Pythium aphanidermatum, Fusarium solani or F. oxysporum. 
In certain embodiments, the array further comprises at least one positive control probe (e.g., a universal probe or a probe that hybridizes to a target sequence found in a class of pathogens, for example, such as a target sequence found in all fungi and/or oomycetes).
In certain embodiments, the at least one positive control probe is selected from SEQ ID NO:48 to SEQ ID NO:51 and SEQ ID NO:305 to SEQ ID NO:308.
In certain embodiments, the array further comprises at least one negative (i.e., internal) control probe (e.g., a probe that is designed not hybridize to any sequence from a target pathogen, such as, for example, a probe that contains nucleotide mismatches as compared to the positive control probe sequence, for example, two mismatched nucleotides).
In certain embodiments, the at least one negative control probe is selected from SEQ ID NO:57, SEQ ID NO:58, and SEQ ID NO:302 to SEQ ID NO:304.



BRIEF DESCRIPTION OF THE FIGURES
FIG. 1. Schematic representation of the five types of oligonucleotide probes used in the diagnostic macroarray. M=monomer (20-24 nt); D=dimer (40-48 nt); DA=dimer with poly-adenine spacer (black boxes) of 10 bases between the two repeats (50-58 nt); M-A20=monomer with a poly-adenine tail (black boxes) of 20 bases (40-44 nt); and M-A10=monomer with a poly-adenine tail (black boxes) of 10 bases (30-34 nt).
FIG. 2. Macroarray design and hybridization results. FIG. 2A, Macroarray design. Specific probes for Pythium aphanidermatum (Pa), Rhizoctonia solani (Rs), Fusarium solani (Fs), and F. oxysporum (Fo, Fox) were spotted in the four shaded regions. Each of the five types of oligonucleotide probes was spotted in a row as follows: row A, DA; B, dimer; C, monomer; D, M-A10; E, M-A20; F (except F8 spotted with monomer), DA; G (except G8 spotted with M-A10), dimer; H (except H8 spotted with M-A20), monomer. Positive controls were spotted in A1, B1, C1, D1, E1, G1, H1, F8, G8, and H8. Internal controls were spotted in A5, B5, C5, D5 and E5. FIG. 2B, C, D and E show macroarray hybridization results with P. aphanidermatum, F. solani, R. solani, and F. oxysporum, respectively.
FIG. 3. Signal intensity comparison of different oligonucleotide probe types. Y-axis represents the mean signal intensity levels as measured with inverted grayscale values. For each probe type (X-axis), there are three independent probes targeting each species (four species tested) except for F. oxysporum which had two probes per probe type, making a total of 11 unique probes for M, D and DA oligonucleotide probe types. Only two species (6 unique probes) were tested for M-A10 and M-A20 probe types. Two isolates were tested for each species except for F. oxysporum, which had only one isolate available. The experiment was conducted twice. Means with the same letter do not differ significantly (Tukey test, P<0.05, n=11 for M, D, and DA whereas n=6 for M-A10 and M-A20).
FIG. 4. Coefficient of variation of hybridization signal intensity among different probe types for the four target species. The coefficient of variation (CV), is defined as the standard deviation divided by the means of the hybridization signals and is used here as a measure of the variation of signal intensity.
FIG. 5. Macroarray results when probes were hybridized with amplified Rhizoctonia solani from 10× serial diluted genomic DNA (10 fg to 1 ag).



DETAILED DESCRIPTION
Disease management can be improved with more rapid and more accurate pathogen detection and identification techniques. As described herein, a macroarray diagnostic technique with enhanced detection sensitivity has been developed. The use of repeat sequence probes (dimers) greatly improves the sensitivity of the macroarray. The dimeric probes reliably detected 0.01 fg target genomic DNA, which is lower than the detection limits of most currently available molecular diagnostic methods, such as the conventional PCR and real-time PCR. Dimer probes also had lower signal variability, thereby increasing the macroarray signal uniformity. This technique is useful for early human, animal or plant disease diagnosis, e.g., when only trace amounts of target microbes are present in a sample. The technique can be adapted and applied to microbial ecological studies and other research areas.
Traditionally, plant diagnosticians use direct observations and/or culturing of pathogens from diseased plant samples to make a diagnosis. These methods are often time consuming and insufficient to identify pathogens to the species level. More recent advancements, such as serology and PCR assays, also have their drawbacks. For instance, immunoassay typing with antibodies has been found to be less specific than DNA-based methods. Quantitative PCR (qPCR), a widely used technology in medical, agriculture, and the food industry, offers an alternative detection platform. However, it is limited in terms of throughput where only one or a few pathogens can be detected in a test reaction (van Doom et al., Appl. Environ. Microbiol., 75, 4185-4193 (2009); Ivnitski et al., Biotechniques 35, 862-869 (2003); Uttamchandani et al., Trends Biotechnol. 27, 53-61 (2009); Lievens et al., FEMS Microbiol. Lett. 223, 113-122 (2003); Lievens et al., Environ. Microbiol. 7, 1698-1710 (2005)). Considering the vast diversity of pathogens, an ideal pathogen detection tool would be characterized by its monitoring capacity for a wide range of pathogen groups as well as by its accuracy and sensitivity (Lee et al., J. Microbiol. Meth. 65, 453-467 (2010)).
DNA diagnostic arrays are another molecular tool that offer a fast, culture-independent alternative for the detection of microbes from field samples (Lievens et al., Phytopathology 95, 1374-1380 (2005); Lievens et al., J. Microbiol. Meth. 80, 76-85; (2010); Zhang et al., Plant Dis. 92, 953-960 (2008); Gilbert et al., J. Cotton Science 12, 165-170 (2008)). The advantage of the array technique is its high throughput compared to other detection methods. Hundreds of different pathogens can be simultaneously detected with one array in one reaction in less than 12 hours. Compared to the glass-based, high-density microarray, the membrane-based macroarray offers a cost-efficient and flexible platform and, therefore has been adopted by many disease diagnosis development projects (Maoka et al., Plant Dis. 94, 1248-1254 (2010)). Moreover, macroarray results can be visualized with an unaided eye, which offers simplicity compared to microarrays. Recently, the application of chromogenic technology in macroarrays has further increased their versatility for use in laboratories or diagnostic labs with limited resources (Abdullahi et al., J. Virol. Methods 160, 90-100 (2009)).
Like other DNA diagnostic array technologies, macroarrays combine the advantage of two molecular biological advancements. First, the growing amount of DNA sequences available in publicly accessible databases (e.g., GenBank) allows for creation of signature probes specific to a species or infra-species target. Second, the high throughput capacity of the array technology permits hundreds of DNA oligomers to be queried simultaneously and produces signals indicative of matches between the oligomer and the query. The macroarray technique has been applied in a variety of areas. For example, in human biology it has been used for identification of different mRNA species present in human ejaculated spermatozoa (Dadoune et al., Mol. Hum. Reprod. 11, 133-140 (2004)) and in diagnosing ovarian cancer in epithelial cells (Chatterjee et al., Cancer Res. 66, 1181-1190 (2006)); while in veterinary science, macroarrays have been used for monitoring the Crimean-Congo Hemorrhagic fever virus, a tickborne zoonotic virus found across Africa, Eastern Europe and Asia (Wolfel et al., J. Clin. Microbiol. 47, 1025-1030 (2009)). In plant pathology, membrane-based DNA arrays have been used for detection, identification, monitoring and quantification of phytopathogenic agents (Fessehaie et al., Phytopathology 93, 262-269 (2003); Sholberg et al., Plant Dis. 89, 1143-1150 (2005)), such as phytopathogenic bacteria on potato, pathogens of apples, and pathogenic viruses and fungi in different host plants. At the infra-species level, DNA diagnostic arrays have been used for identification of races and biotypes of Fusarium oxysporum f. sp. vasinfectum on cotton and the detection of members of the F. solani species complex in solanaceous crops (Zhang et al., Plant Dis. 91, 1612-1620 (2007)).
Probe design is the first step in the development of a diagnostic array. Parameters such as probe length and annealing temperatures (or GC content) play a role in array performance that have great impact on the fidelity of the assay, particularly with regard to the level of specificity and sensitivity attained (Barad et al., Genome Res. 14, 2486-2494 (2004); Goff et al., RNA Biology 2, E9-E16 (2005); Loy et al., Clin. Chim. Acta 363, 106-119 (2006)). If probes are not optimized for specificity, arrays may generate false positives due to cross-hybridization to similar sequences. On the other hand, high stringency often results in reduced signal intensity and may lead to false negatives. The occurrence of false positives and negatives is problematic because it is difficult to envisage whether a probe will attach efficiently to its target sequence and yield a good hybridization signal based on the sequence information alone (Li et al., Bioinformatics 17, 1067-1076 (2001); Chou et al., Nucleic Acids Res. 32, e99 (2004)). The design of effective probes is a challenge especially in related species where there is a high degree of sequence similarities. Scientists have therefore resulted to engineering redundancy into the DNA array's systems to improve accuracy and analytical power simply by over-representation. That is, using multiple probes per target to achieve reliable and accurate detection.
A number of studies have been able to achieve high levels of specificity with DNA arrays, but sensitivity has remained elusive (Wong et al., Genome Biol. 8, R93 (2007)), which made it difficult to detect species that were present at very low concentrations.
Increasing probe length can increase the array sensitivity, but specificity is often sacrificed. However, the impact of doubling (dimer) or tripling (trimer) a short probe sequence (about 20 nt monomer) to maintain the array specificity has not been addressed.
The objectives of this study were to develop a novel technical approach that could increase the sensitivity of a macroarray to enhance its early pathogen detection power, and which could maintain the macroarray detection specificity to ensure accurate pathogen identification. In this study, we designed and compared the performance of monomers, monomers with a ten-adenine tail (M-A10), monomers with 20 adenine tail (M-A20), dimers, and dimers with a ten-adenine spacer (DA), using a membrane-based macroarray platform (FIG. 1). It was hypothesized that (1) Dimeric oligonucleotide probes would be more sensitive than monomeric probes, (2) Poly-A spacer and tails would increase sensitivity of the probes, and that (3) Monomeric and dimeric probes would have equal specificity. Probes tested in this study were based on four important microbial pathogens of cereals, turfgrass and other plants—Rhizoctonia solani (basidiomycete), Pythium aphanidermatum (oomycete), Fusarium solani (ascomycete) and F. oxysporum (ascomycete) that cause brown patch, Pythium blight, root and vascular diseases, respectively.
Rapid and early diagnosis of microbe-causing diseases requires a technique capable of detecting low quantity of causal agents from the natural host environment. Accordingly, described herein is an improved macroarray detection technique that provides enhanced and consistent signals of detection with small reductions in specificity. The tandem-repeat dimeric probes (40-48 nt) had significantly higher sensitivity and lower signal variability compared to the monomers (20-24 nt).
The diagnostic array technique for microbe detection demands both high sensitivity and specificity. While developing macroarrays for the detection of solanaceous plant pathogens, Zhang et al. (2007) optimized the hybridization temperature to reduce cross-hybridization. However, this improvement in specificity came with a considerable sacrifice in signal intensity, which is also determined by the probe length. Long sequence probes can decrease the array specificity. The hypothesis that dimeric probes containing two short identical sequences would enhance the DNA array sensitivity without sacrificing the specificity was tested. However, two monomers in tandem might interfere with hybridization of targets to the two matching sequences. Thus, the possibility that addition of a spacer in between may facilitate the binding of long target DNA fragments to the probes without unnecessary tangling was tested. However, the results showed that overall, dimers with poly-A spacer did not produce stronger signals than the dimers. This suggests that the proximity of two monomers to each other does not have a significant effect on hybridization. Previous work has shown that addition of spacers can have a large effect on hybridization signals for 15-30 mer oligonucleotide probes (Shchepinov et al., Nucleic Acids Res. 25, 1155-1161 (1997); Southern et al., Nature Genet., 21, 5-9 (1999); Guo et al., Nat. Biotechnol. 15, 331-335 (1997)).
To assess the reproducibility of the results, eleven sets of probes for DA, D, and M types (two to three independent sets for each of the four species) in addition to the controls were examined in this study. For the diagnostic arrays, multiple probes for each target species also ensures positive detection of genetically diverse target species. Random signal variation (noise) and the systematic deviation of the measurement from the true signal due to probe specific or other confounding technical effects can interfere with the results. Signal variation, expressed as a coefficient of variation (CV), showed that dimer probes had lowest variation while the monomers showed wide variability. Based on the statistical principles of sampling, the smaller the CV for the hybridization signals, the more reliable or reproducible the results are (Chou et al., 2004). This implies that fewer probes are needed when using dimer probes.
Although dimeric probes provided a low measurement variation and superior signal intensity, some dimeric probes tested here were relatively poor in discriminating sequences with high level of similarity (especially 1 nt differences). Cross-homology is a predicator of cross-hybridization. The concept of using dimers as probes does not change the level of similarity compared to the monomers. Therefore, the cross-hybridization observed maybe a consequence of other factors such as the binding energy accruing from longer probes. Longer probes typically have higher binding energy than shorter probes. Cross-hybridization observed here was only limited to members of the same genus and occurred in highly similar sequences where the mismatch base was located near the end or in a chain of the same base. This should be preventable in future array probe designs by avoiding such regions of a gene. These results showed that in all cases, the dimeric probes were able to distinguish strains that differed from the target by 3 or more nucleotides. Full discrimination also achieved for most cases of 2-nucleotide mismatches and one case of single nucleotide match. Therefore, with improved probe design strategy as described, the dimer array system is expected to distinguish between closely related pathogens, at species and infra species levels, such as race and subspecies.
Even though this macroarray system displayed some false-positives, it was remarkable in detecting low quantities of pathogen DNA. The dimer probes were able to detect as low as 0.1 pg target DNA in raw or mixed biological samples with plant extract despite the possibility of PCR bias in amplification. The dimers also reliably detected 0.01 fg target DNA from pure cultures on the array, while gel electrophoresis required a thousand fold more DNA for a positive detection. Assessment of low concentrations of target DNA that is only present in few plant cells can be very elusive. Understanding pathogen biology is of paramount importance in disease diagnosis, since some pathogens are localized in certain parts of the tissue, while others are systemic in nature or cause symptoms in advance of tissue pathogen ingress. Visualization of pathogen structures using dissecting microscope to locate tissue with signs of the disease followed by targeted isolation of DNA from these tissues may improve the power of molecular detection.
In summary, reported herein is the finding that dimeric probes (e.g., 40-48 nt) enhance macroarray performance. The optimized dimer macroarray system demonstrated significantly higher sensitivity and consistency than the conventional monomer oligonucleotide arrays. Its detection sensitivity is also higher than many other currently available molecular diagnostic methods, such as PCR and real-time PCR. Moreover, this method is faster (less than 12 hours) than the traditional culture-based diagnostic method, which often takes days or even weeks. Therefore, this technique should be useful for early disease diagnosis when only trace amounts of target microbes are present in a sample. These findings should aid in the development of a multiplex diagnostic macroarray system to facilitate early disease diagnosis and management. The technique also can be adapted and applied to microbial ecological studies and other research areas.
The term “nucleic acid” refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form, made of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine or pyrimidine. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues.
The term “nucleotide sequence” refers to a polymer of DNA or RNA which can be single-stranded or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers. The terms “nucleic acid,” “nucleic acid molecule,” or “polynucleotide” are used interchangeably.
Certain embodiments of the invention encompass isolated or substantially purified nucleic acid compositions. In the context of the present invention, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature. An isolated DNA molecule or RNA molecule may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. For example, an “isolated” or “purified” nucleic acid molecule is substantially free of other cellular material or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
The following terms are used to describe the sequence relationships between two or more nucleotide sequences: (a) “reference sequence,” (b) “comparison window,” (c) “sequence identity,” (d) “percentage of sequence identity,” and (e) “substantial identity.”
(a) As used herein, “reference sequence” is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
(b) As used herein, “comparison window” makes reference to a contiguous and specified segment of a polynucleotide sequence, wherein the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Generally, the comparison window is at least 20 contiguous nucleotides in length, and optionally can be 30, 40, 50, 100, or longer. Those of skill in the art understand that to avoid a high similarity to a reference sequence due to inclusion of gaps in the polynucleotide sequence a gap penalty is typically introduced and is subtracted from the number of matches.
Methods of alignment of sequences for comparison are well-known in the art. Thus, the determination of percent identity between any two sequences can be accomplished using a mathematical algorithm. Non-limiting examples of such mathematical algorithms are the algorithm of Myers and Miller (Myers and Miller, CABIOS, 4, 11 (1988)); the local homology algorithm of Smith et al. (Smith et al., Adv. Appl. Math., 2, 482 (1981)); the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)); the search-for-similarity-method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85, 2444 (1988)); the algorithm of Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87, 2264 (1990)), modified as in Karlin and Altschul (Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90, 5873 (1993)).
Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters. The CLUSTAL program is well described by Higgins et al. (Higgins et al., CABIOS, 5, 151 (1989)); Corpet et al. (Corpet et al., Nucl. Acids Res., 16, 10881 (1988)); Huang et al. (Huang et al., CABIOS, 8, 155 (1992)); and Pearson et al. (Pearson et al., Meth. Mol. Biol., 24, 307 (1994)). The ALIGN program is based on the algorithm of Myers and Miller, supra. The BLAST programs of Altschul et al. (Altschul et al., JMB, 215, 403 (1990)) are based on the algorithm of Karlin and Altschul supra.
Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is less than about 0.1, less than about 0.01, or even less than about 0.001.
To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, the default parameters of the respective programs (e.g., BLASTN for nucleotide sequences, BLASTX for proteins) can be used. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix. Alignment may also be performed manually by inspection.
For purposes of the present invention, comparison of nucleotide sequences for determination of percent sequence identity to the promoter sequences disclosed herein may be made using the BlastN program (version 1.4.7 or later) with its default parameters or any equivalent program. By “equivalent program” is intended any sequence comparison program that, for any two sequences in question, generates an alignment having identical nucleotide or amino acid residue matches and an identical percent sequence identity when compared to the corresponding alignment generated by the program.
(c) As used herein, “sequence identity” or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to a specified percentage of residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window, as measured by sequence comparison algorithms or by visual inspection. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.” Means for making this adjustment are well known to those of skill in the art. Typically this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., as implemented in the program PC/GENE (Intelligenetics, Mountain View, Calif.).
(d) As used herein, “percentage of sequence identity” means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
(e)(i) The term “substantial identity” of polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even at least 95%, 96%, 97%, 98%, or 99% sequence identity, compared to a reference sequence using one of the alignment programs described using standard parameters. One of skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by two nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning, and the like. Substantial identity of amino acid sequences for these purposes normally means sequence identity of at least 70%, 80%, 90%, or even at least 95%.
Another indication that nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C., depending upon the desired degree of stringency as otherwise qualified herein. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides they encode are substantially identical. This may occur, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. One indication that two nucleic acid sequences are substantially identical is when the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
(e)(ii) The term “substantial identity” in the context of a peptide indicates that a peptide comprises a sequence with at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, or 94%, or even 95%, 96%, 97%, 98% or 99%, sequence identity to the reference sequence over a specified comparison window. In certain embodiments, optimal alignment is conducted using the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, JMB, 48, 443 (1970)). An indication that two peptide sequences are substantially identical is that one peptide is immunologically reactive with antibodies raised against the second peptide. Thus, a peptide is substantially identical to a second peptide, for example, where the two peptides differ only by a conservative substitution. Thus, certain embodiments of the invention provide nucleic acid molecules that are substantially identical to the nucleic acid molecules described herein.
For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
As noted above, another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions. The phrase “hybridizing specifically to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. “Bind(s) substantially” refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accommodated by reducing the stringency of the hybridization media to achieve the desired detection of the target nucleic acid sequence.
“Stringent hybridization conditions” and “stringent hybridization wash conditions” in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence dependent, and are different under different environmental parameters. Longer sequences hybridize specifically at higher temperatures. The thermal melting point (Tm) is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984); Tm 81.5° C.+16.6 (log M)+0.41 (% GC)-0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with >90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the Tm for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the Tm; moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the Tm; low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the Tm. Using the equation, hybridization and wash compositions, and desired temperature, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a temperature of less than 45° C. (aqueous solution) or 32° C. (formamide solution), the SSC concentration is increased so that a higher temperature can be used. Generally, highly stringent hybridization and wash conditions are selected to be about 5° C. lower than the Tm for the specific sequence at a defined ionic strength and pH.
An example of highly stringent wash conditions is 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent wash conditions is a 0.2×SSC wash at 65° C. for 15 minutes. Often, a high stringency wash is preceded by a low stringency wash to remove background probe signal. An example medium stringency wash for a duplex of, e.g., more than 100 nucleotides, is 1×SSC at 45° C. for 15 minutes. For short nucleotide sequences (e.g., about 10 to 50 nucleotides), stringent conditions typically involve salt concentrations of less than about 1.5 M, less than about 0.01 to 1.0 M, Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is typically at least about 30° C. and at least about 60° C. for long probes (e.g., >50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. In general, a signal to noise ratio of 2× (or higher) than that observed for an unrelated probe in the particular hybridization assay indicates detection of a specific hybridization. Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the proteins that they encode are substantially identical. This occurs, e.g., when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code.
Very stringent conditions are selected to be equal to the Tm for a particular probe. An example of stringent conditions for hybridization of complementary nucleic acids that have more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide, e.g., hybridization in 50% formamide, 1 M NaC1, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaC1, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.
In addition to the chemical optimization of stringency conditions, analytical models and algorithms can be applied to hybridization data-sets (e.g. microarray data) to improve stringency.
Certain embodiments of the invention will now be illustrated by the following non-limiting Examples.
EXAMPLE 1
Dimeric Oligonucleotide Probes Enhance Diagnostic Macroarray Performance 
Disease management would be improved with more rapid and more accurate pathogen detection and identification techniques. Described herein is a macroarray diagnostic technique with enhanced detection sensitivity with only small reduction in specificity. With probes designed based on the internal transcribed spacer sequences of the rRNA genes of fungal and oomycete strains, a macroarray was produced that included five types of oligonucleotide probes: monomers (20-24 nt), dimers (40-48 nt), dimers with a poly-A spacer of 10 bases between the two repeats (50-58 nt), monomers with a poly-A tail of 10 (30-34 nt) and 20 (40-44 nt) bases. The use of repeat sequence probes (dimers) greatly improved the sensitivity of the macroarray. The dimeric probes could reliably detect 0.01 fg target genomic DNA, which is lower than the detection limits of most currently available molecular diagnostic methods, such as the conventional PCR and real-time PCR. Dimer probes also had lower signal variability, thereby increasing the macroarray signal uniformity. However, in a few cases, specificity was reduced in the dimer probes. Cross-hybridization occurred in highly similar sequences where the mismatched base was located near the end or in a chain of the same base, but this should be prevented in future array probe design.
Probe Sensitivity
Dimers and DA had significantly higher sensitivity than those of the M, M-A10 and M-A20 (FIG. 3). To test whether the monomers were being outcompeted in the race for hybridization targets, monomers were printed on a separate membrane. Results showed that there was no significant difference in signal intensities between monomers printed on a separate array and monomers printed in same array with dimers (Table 5).
Signal Uniformity
Signal intensities of monomeric probes were most variable compared to the other types of probes (FIG. 2, FIG. 4). The signal intensities for monomers were so diverse that F. oxysporum had one probe with an inverted gray value of 89 while the other was showing only 4. On the same array, dimers and dimers with poly-A spacer derived from those monomers had lesser inverted gray value disparity (117-108 and 117-114, respectively). The coefficient of variation (CV), defined here as the standard deviation divided by the means of the hybridization signals was used as a measure of the variation of signal intensity (Chou et al., 2004; FIG. 4). Overall, the dimers displayed the lowest variability, followed by the dimers with poly-A and then the monomers M-A20 and M-A10. Monomers alone had the greatest variability.
Limit of Detection and Validation
The dimer probes could reliably detect up to 0.01 fg genomic DNA, which is a thousand times lower than using PCR product visualization with gel electrophoresis (FIG. 5). A simulation based on a condition where the pathogen DNA was serially diluted while holding the host grass DNA at 1 ng showed that dimeric probes could detect target DNA at all levels tested (Table 3), including 0.1 pg at a ratio of 1:104 pathogen to host DNA for R. solani and P. aphanidermatum. Mixing the DNA of the target species, commonly found co-inhabiting fungal species and the host plant did not interfere with the hybridization reactions for all reactions tested (Table 3). The macroarray was also successfully validated with target species infected plant or soil materials. The array detection and identification results matched with the identification based on traditional microscopic observation, culture isolation and ITS DNA sequence (Table 1).
Array Specificity
There was no cross hybridization to P. aphanidermatum and R. solani probes from any of the non-target isolates. Cross-hybridization was observed in three sets of dimers and DA probes (Table 6) when reacting with non-target species that had one or two nucleotide sequence mismatches (Table 4). The mismatches between the cross-hybridized probes and the corresponding non-target sequences were located either in chain of A or G (F. solani #F19/probe Fs6 and F. equiseti #S2/probe Fo1, Tables 4) or near the end of the probe sequence (F. solani #F3/probe Fs13). The cross-hybridization signal intensity values were 44% or lower compared to the perfect-match signals. False negatives were only observed in a monomer probe for P. aphanidermatum (Table 6).
Materials and Methods
Isolates
Test isolates used in this study are listed in Table 1. The identity of the target species, P. aphanidermatum and R. solani, was confirmed by morphology and the internal transcribed spacer sequences of the rRNA genes (ITS), while the Fusarium species were characterized in another study by partial sequences of translation elongation factor-1α (EF-1α), ITS, and β-tubulin (TUB) genes. In addition, non-target species of Pythium, Rhizoctonia, and Fusarium were used to test the array specificity. Three common co-inhabiting fungi in turfgrass soil, Curvularia trifolii, Trichoderma vixens, and Mortierella elongata were also included in the study for validation and cross-reaction tests.







TABLE 1







Microbial, host species and substrate used for testing and validating the macroarray in this study















GenBank


Microbial/host species
Collection ID
Host/substrate
Origin
accession





Fungi-Zygomycota






Mortierella elongata
#141
Poa annua
Denville, NJ
—


Fungi-Ascomycota


Curvularia trifolii
#185
Agrostis sp.
New Brunswick, NJ
—


Fusarium equisetti
#S2
turfgrass soil
New Brunswick, NJ
—


F. oxysporum
#F2 (NRRL 54168)
Lilium longiflorum bulbs
New Brunswick, NJ
HQ379648


F. oxysporum
#S4
turfgrass soil
New Brunswick, NJ
—


F. solani
#F3 (NRRL 54169)
Lilium longiflorum bulbs
New Brunswick, NJ
HQ379661


F. solani
#F19 (NRRL 54185)
Lilium longiflorum bulbs
New Brunswick, NJ
HQ379663


Trichoderma virens
#126-L
Agrostis stolonifera
New Brunswick, NJ
—


Fungi-Basidiomycota


Rhizoctonia solani
#RH 20
unknown
State College, PA
—


R. solani
#98
Lolium multiflorum
New Brunswick, NJ
—


Waitea circinata
#10
Agrostis sp.
NJ
—


var. circinata


W. circinata
#158
Agrostis sp./Poa sp.
Bedminster, NJ
HQ166071


var. circinata


W. circinata var. zeae
#1
Poa annua
New Brunswick, NJ
—


Stramenopiles-Oomycota


Pythium aphanidermatum
#60
Festuca arundinacea
NJ
—


P. aphanidermatum
#99
Lolium multiflorum
New Brunswick, NJ
—


P. rostrum
#123p
Agrostis sp.
New Brunswick, NJ
—


P. torulosum
#122
Poa annua
New Brunswick, NJ
—


P. volutum
#124p-1
poa annua
New Brunswick, NJ
—


Host/substrate


—a
#199
Agrostis stolonifera
New Brunswick, NJ
—


—b
NA
Poa annua
New Brunswick, NJ
—


—c
#176
Poa annua
New Brunswick, NJ
—


—d
NA
Soil
New Brunswick, NJ
—





NRRL = Agricultural Research Service Culture Collection (NCAUR, Peoria, IL).


aAgrostis stolonifera with symptoms of brown patch.


bAsymptomatic, disease free Poa annua from greenhouse.


cPoa annua with symptoms of Pythium blight.


Soil substrates used were those associated with P. aphanidermatum and R. solani.


NA = Not available.






DNA Extraction, Amplification, Purification and Sequencing
Genomic DNA from all microbial isolates was extracted from 1- to 2-week-old cultures growing on PDA plates (Difco laboratories, Detroit, Mich.) using the UltraClean Soil DNA kit (MoBio Laboratories Inc., Solana Beach, Calif., USA) or DNeasy Plant Mini kit (Qiagen Inc., Valencia, Calif., USA), following the manufacturer's protocol. The extracted genomic DNA was quantified using a NanoVue spectrophotometer (GE Healthcare Bio-Sciences Corp., Piscataway, N.J., USA) and diluted to 5 ng/μl before PCR. The ITS region was amplified with primers ITS1 and ITS4 (White et al., In: Innis et al. (Eds.), PCR Protocols: A Guide to Methods and Applications. Academic Press, New York, pp. 315-322 (1990)). PCR was carried out in a 25 μl reaction volume containing 3 μl (15 ng) genomic DNA, 1×PCR buffer (Applied Biosystems, Foster City, Calif., USA), 1.5 mM MgCl2, 200 μM each of the dNTPs, 0.5 μM of each forward and reverse primers, and 0.5 U Taq polymerase. The thermal cycling parameters were 95° C. for 5 min; 35 cycles of 95° C. for 1 min, 56° C. for 1 min., and 72° C. for 1 min; followed by 10 min at 72° C. PCR products were purified according to the manufacturer's protocol using the QIAquick PCR Purification Kit (Qiagen), quantified using NanoVue spectrophotometer and sequenced when identity of the isolates needed to be confirmed. Sequencing of the purified ITS PCR products was run on an Applied Biosystems 3730xl sequencer by GENEWIZ (GENEWIZ, Inc., South Plainfield, N.J., USA).
Oligonucleotide Probe Design and Array Development
Two sets of probes, dimers and dimers with a poly-A10 spacer of 10 bases for the four target species and the controls (Table 2) were designed based in part on 20-24 nt oligomer probes previously designed and validated by Lievens et al. (2003), Saiki et al., Proc. Natl. Acad. Sci. USA 86, 6230-6234. (1989), and Zhang et al., (2007, 2008). Accordingly, certain embodiments of the invention are directed to these probes. M-A10 and M-A20 were designed for P. aphanidermatum and F. solani only (Table 2), since we were more interested with the effect of duplexing but at the same time we wanted to disqualify sequence length as the only factor contributing to the enhanced sensitivity. The performance of the macroarray that contained the five types of probes was tested against the macroarray that contained only the monomeric probes.
The macroarray development followed the procedure described by Zhang et al. (2007). Briefly, 20 μmol of each detector oligonucleotide probe was spotted onto Hybond N+ nylon membranes (GE Healthcare Bio-Sciences Corp., Piscataway, N.J., USA) in quadruplicate using a 96-pin tool (V&P Scientific Inc., San Diego, Calif., USA). Three types of controls were also spotted. First, the positive controls included the ITS4 primer, which is a universal primer for both fungi and oomycetes, and ITS2, which is a fungal universal primer (Table 2). Second, internal controls that differed from ITS2 at two bases were also spotted on the membrane. Negative controls were sterile water and the spotting buffer. The positive and internal controls also constituted dimers, DA, M-A10 and M-A20. The spotted membranes were air dried for 10 min and then fixed by UV exposure at 240 mJ/cm2. After incubation in a 0.5% sodium dodecyl sulfate (SDS) solution at 60° C. for an hour, membranes were rinsed in 100 mM Tris (pH 8.0) for 5 min, and kept moist at 4° C. until used. This last step was also used for stripping the array.







TABLE 2







Probe name, type, sequence, length and targeted species used in this study.












Probe
Probe


Probe



name
typea
Probe sequence
Referenceb
Length
Probe target





Specific







target







probe







Pa1
M
GGAGAGAGATGGCAGAATGTGAG
Saiki et
23
P. aphanidermatum




(SEQ ID NO: 1)
al. 1989




 


Pa2
M
GGGAGAGAGATGGCAGAATGTGAG
Saiki et
24
P. aphanidermatum




(SEQ ID NO: 2)
al. 1989




 


Pa3
M
GAGGTGTACCTGAATTGTGTGAGG
Saiki et
24
P. aphanidermatum




(SEQ ID NO: 3)
al. 1989




 


Pa1R
D
GGAGAGAGATGGCAGAATGTGAGGGAGAGAGATGGCAGAATGTGAG

46
P. aphanidermatum




(SEQ ID NO: 4)





 


Pa2R
D
GGGAGAGAGATGGCAGAATGTGAGGGGAGAGAGATGGCAGAATGTG

48
P. aphanidermatum




AG







(SEQ ID NO: 5)





 


Pa3R
D
GAGGTGTACCTGAATTGTGTGAGGGAGGTGTACCTGAATTGTGTGA

48
P. aphanidermatum




GG







(SEQ ID NO: 6)





 


Pa1R-A
DA
GGAGAGAGATGGCAGAATGTGAGAAAAAAAAAAGGAGAGAGATGGC

56
P. aphanidermatum




AGAATGTGAG







(SEQ ID NO: 7)





 


Pa2R-A
DA
GGGAGAGAGATGGCAGAATGTGAGAAAAAAAAAAGGGAGAGAGATG

58
P. aphanidermatum




GCAGAATGTGAG







(SEQ ID NO: 8)





 


Pa3R-A
DA
GAGGTGTACCTGAATTGTGTGAGGAAAAAAAAAAGAGGTGTACCTG

58
P. aphanidermatum




AATTGTGTGAGG







(SEQ ID NO: 9)





 


Pa1-A10
M-A10
GGAGAGAGATGGCAGAATGTGAGAAAAAAAAAA

33
P. aphanidermatum




(SEQ ID NO: 10)





 


Pa2-A10
M-A10
GGGAGAGAGATGGCAGAATGTGAGAAAAAAAAAA

34
P. aphanidermatum




(SEQ ID NO: 11)





 


Pa3-A10
M-A10
GAGGTGTACCTGAATTGTGTGAGGAAAAAAAAAA

34
P. aphanidermatum




(SEQ ID NO: 12)





 


Pa1-A20
M-A20
GGAGAGAGATGGCAGAATGTGAGAAAAAAAAAAAAAAAAAAAA

43
P. aphanidermatum




(SEQ ID NO: 13)





 


Pa1-A20
M-A20
GGGAGAGAGATGGCAGAATGTGAGAAAAAAAAAAAAAAAAAAAA

44
P. aphanidermatum




(SEQ ID NO: 14)





 


Pa3-A20
M-A20
GAGGTGTACCTGAATTGTGTGAGGAAAAAAAAAAAAAAAAAAAA

44
P. aphanidermatum




(SEQ ID NO: 15)





 


Rs2
M
CAGTGTTATGCTTGGTTCCACTC
Zhang et
23
R solani




(SEQ ID NO: 16)
al. 2008




 


Rs3
M
TGTTGAAACTTAGTATTAGATGCGT
Zhang et
23
R. solani




(SEQ ID NO: 17)
al. 2008




 


Rs4
M
GAGTGGAACCAAGCATAACACTG
Zhang et
23
R. solani




(SEQ ID NO: 18)
al. 2008




 


Rs2R
D
CAGTGTTATGCTTGGTTCCACTCCAGTGTTATGCTTGGTTCCACTC

46
R. solani




(SEQ ID NO: 19)





 


Rs3R
D
TGTTGAAACTTAGTATTAGATGCGTTGTTGAAACTTAGTATTAGAT

46
R. solani




GCGT







(SEQ ID NO: 20)





 


Rs4R
D
GAGTGGAACCAAGCATAACACTGGAGTGGAACCAAGCATAACACTG

46
R. solani




(SEQ ID NO: 21)





 


Rs2R-A
DA
CAGTGTTATGCTTGGTTCCACTCAAAAAAAAAACAGTGTTATGCTT

56
R. solani




GGTTCCACTC







(SEQ ID NO: 22)





 


Rs3R-A
DA
TGTTGAAACTTAGTATTAGATGCGTAAAAAAAAAATGTTGAAACTT

56
R. solani




AGTATTAGATGCGT







(SEQ ID NO: 23)





 


Rs4R-A
DA
GAGTGGAACCAAGCATAACACTGAAAAAAAAAAGAGTGGAACCAAG

56
R. solani




CATAACACTG







(SEQ ID NO: 24)





 


Fs4
M
TCGCGTAGTAGCTAACACCTCGC
Zhang et
23
F. solani




(SEQ ID NO: 25)
al. 2007




 


Fs6
M
CCTGTGAACATACCTAAACGTTG
Zhang et
23
F. solani




(SEQ ID NO: 26)
al. 2007




 


Fs13
M
TTATACAACTCATCAACCCTGTGA
Zhang et
24
F. solani




(SEQ ID NO: 27)
al. 2007




 


Fs4R
D
TCGCGTAGTAGCTAACACCTCGCTCGCGTAGTAGCTAACACCTCGC

46
F. solani




(SEQ ID NO: 28)





 


Fs6R
D
CCTGTGAACATACCTAAACGTTGCCTGTGAACATACCTAAACGTTG

46
E solani




(SEQ ID NO: 29)





 


Fs13R
D
TTATACAACTCATCAACCCTGTGATTATACAACTCATCAACCCTGT

48
F. solani




GA







(SEQ ID NO: 30)





 


Fs4R-A
DA
TCGCGTAGTAGCTAACACCTCGCAAAAAAAAAATCGCGTAGTAGCT

56
F. solani




AACACCTCGC







(SEQ ID NO: 31)





 


Fs6R-A
DA
CCTGTGAACATACCTAAACGTTGAAAAAAAAAACCTGTGAACATAC

56
F. solani




CTAAACGTTG







(SEQ ID NO: 32)





 


Fs13R-A
DA
TTATACAACTCATCAACCCTGTGAAAAAAAAAAATTATACAACTCA

58
F. solani




TCAACCCTGTGA







(SEQ ID NO: 33)





 


Fs4-A10
M-A10
TCGCGTAGTAGCTAACACCTCGCAAAAAAAAAA

33
F. solani




(SEQ ID NO: 34)





 


Fs6-A10
M-A10
CCTGTGAACATACCTAAACGTTGAAAAAAAAAA

33
F. solani




(SEQ ID NO: 35)





 


Fs13-A10
M-A10
TTATACAACTCATCAACCCTGTGAAAAAAAAAAA

34
F solani




(SEQ ID NO: 36)





 


Fs4-A20
M-A20
TCGCGTAGTAGCTAACACCTCGCAAAAAAAAAAAAAAAAAAAA

43
F. solani




(SEQ ID NO: 37)





 


Fs6-A20
M-A20
CCTGTGAACATACCTAAACGTTGAAAAAAAAAAAAAAAAAAAA

43
F. solani




(SEQ ID NO: 38)





 


Fs13-A20
M-A20
TTATACAACTCATCAACCCTGTGAAAAAAAAAAAAAAAAAAAAA

44
F. solani




(SEQ ID NO: 39)





 


Fo1
M
CGTTCCTCAAATTGATTGGCGGTC
Zhang et
24
F. oxysporum




(SEQ ID N0: 40)
al. 2008




 


Fox2
M
GTTGGGACTCGCGTTAATTCG
Lievens et
21
F. oxysporum




(SEQ ID N0: 41)
al. 2003




 


Fo1R
D
CGTTCCTCAAATTGATTGGCGGTCCGTTCCTCAAATTGATTGGCGG

48
F. oxysporum




TC







(SEQ ID NO: 42)





 


Fox2R
D
GTTGGGACTCGCGTTAATTCGGTTGGGACTCGCGTTAATTCG

42
F. oxysporum




(SEQ ID NO: 43)





 


Fox2-A
DA
CGTTCCTCAAATTGATTGGCGGTCAAAAAAAAAACGTTCCTCAAAT

58
F. oxysporum




TGATTGGCGGTC







(SEQ ID NO: 44)





 


Fo1R-A
DA
GTTGGGACTCGCGTTAATTCGAAAAAAAAAAGTTGGGACTCGCGTT

52
F. oxysporum




AATTCG







(SEQ ID NO: 45)





 


Positive







control







Probe







ITS2
M
GCTGCGTTCTTCATCGATGC
White et
20
Fungi




(SEQ ID NO: 46)
al. 1990




 


ITS4
M
TCCTCCGCTTATTGATATGC
White et
20
Fungi & oomycete




(SEQ ID NO: 47)
al. 1990




 


ITS2R
D
GCTGCGTTCTTCATCGATGCGCTGCGTTCTTCATCGATGC

40
Fungi




(SEQ ID NO: 48)





 


ITS4R
D
TCCTCCGCTTATTGATATGCTCCTCCGCTTATTGATATGC

40
Fungi & oomycete




(SEQ ID NO: 49)





 


 


ITS2R-A
DA
GCTGCGTTCTTCATCGATGCAAAAAAAAAAGCTGCGTTCTTCATCG

50
Fungi




ATGC







(SEQ ID NO: 50)





 


ITS4R-A
DA
TCCTCCGCTTATTGATATGCAAAAAAAAAATCCTCCGCTTATTGAT

50
Fungi & oomycete




ATGC







(SEQ ID NO: 51)





 


ITS2-A10
M-A10
GCTGCGTTCTTCATCGATGCAAAAAAAAAA

30
Fungi




(SEQ ID NO: 52)





 


ITS4-A10
M-A10
TCCTCCGCTTATTGATATGCAAAAAAAAAA

30
Fungi & oomycete




(SEQ ID NO: 53)





 


ITS2-A20
M-A20
GCTGCGTTCTTCATCGATGCAAAAAAAAAAAAAAAAAAAA

40
Fungi




(SEQ ID NO: 54)





 


ITS4-A20
M-A20
TCCTCCGCTTATTGATATGCAAAAAAAAAAAAAAAAAAAA

40
Fungi & oomycete




(SEQ ID NO: 55)





 


Internal







control







Probe







ITS2-2-1
M
GCTGCGTTGATCATCGATGC
Zhang et
20
None




(SEQ ID NO: 56)
al. 2008




 


ITS2-2-1R
D
GCTGCGTTGATCATCGATGCGCTGCGTTGATCATCGATGC

40
None




(SEQ ID NO: 57)





 


ITS2-2-
DA
GCTGCGTTGATCATCGATGCAAAAAAAAAAGCTGCGTTGATCATCG

50
None


1R-A

ATGC







(SEQ ID NO: 58)





 


ITS2-2-
M-A10
GCTGCGTTGATCATCGATGCAAAAAAAAAA

30
None


1-A10

(SEQ ID NO: 59)





 


ITS2-2-
M-A20
GCTGCGTTGATCATCGATGCAAAAAAAAAAAAAAAAAAAA

40
None


1-A20

(SEQ ID NO: 60)








aD = dimer, DA = dimer with 10 adenine nucleotides(poly-A) spacer, M = monomer, M-A10 = monomer with 10 adenine nucleotides


(poly-A10) tail and M-A20 = monomer with 20 adenine nucleotides(poly-A20) tail.


bReference for literature citations. Oligonucleotide probes that do not have a reference were modifications from referenced probes by


the authors for this study.






Hybridization

Hybridization was carried out as described previously (Zhang et al., 2007, 2008). The test ITS amplicons from either target or non-target materials were labeled and hybridized using the Gene Images AlkPhos Direct Labeling (GE Healthcare Bio-Sciences Corp., Piscataway, N.J., USA) and Detection System with CDP-Star (Topix Inc., Bedford, Mass., USA). Before use, the arrays were pre-hybridized at 55° C. for 15 min and hybridized with 100 ng (10 μl of 10 m/μl) of labeled ITS amplicon at 55° C. for 2 h. After two primary washes and two secondary washes, the detection reagent was added to the array to react for an hour, followed by 30 min of film exposure. Chemiluminescence was detected using Kodak Biomax Light film. Developed films were scanned by an Aficio MP C6000 Color Copier/Scanner (Ricoh Americas Corporation, West Caldwell, N.J., USA) and read with ImageJ 1.33u (National Institutes of Health, MD, USA).
Array Sensitivity
The signal intensity for hybridization was measured as the average inverted gray value for the quadruplicate spots for each detector oligonucleotide on the array after the background gray values were subtracted. Since in 8-bit grayscale images, the darkest picture corresponds to the lowest value, each value was inverted by subtracting its reading from 255 (i.e. inverted gray value=255-gray value readings). Each experiment was conducted at least twice. The signal intensity of the monomers was compared vis-à-vis those of the dimer, DA and where applicable, M-A10 and M-A20 for each isolate.
Two isolates, R. solani (#98) and P. aphanidermatum (#99), were used to determine the detection limit of the PCR-coupled macroarray method. The genomic DNA of #98 and #99 was 10× serially diluted from 1 ng/μl to 1×10−10 ng/μl prior to a standard ITS PCR. Following ITS amplification, hybridization was conducted as described above. Ten μl of purified ITS PCR product was used for hybridization and gel electrophoresis. The experiments were conducted twice. To determine whether DNA from the host and other co-inhabiting fungi would interfere with the target species detection, the target DNA was 10× serially diluted (from 1 ng/μl to 1×10−4 ng/μl) and mixed with 1 ng of plant DNA. Two ul of the mixture were used for PCR amplification in a 25 μl reaction mix (Table 3). Plant DNA was derived from clean greenhouse grown Poa annua with no history of disease infection. Serially diluted genomic DNA of the target species (from 1 ng/μl to 1×10−2 ng/μl of #98 or #99) was mixed in same ratio with the serially diluted genomic DNA from three common turfgrass-associated fungi, C. trifolii, T. virens, and M. elongata (Table 3). PCR amplification and hybridization were done as explained above. Control experiments were done to test if the observed results were due to skewed amplification by PCR, by mixing 2.5 ng of purified PCR products of each of the test sample (P. annua, C. trifolii, T. virens, M. elongata, and the target species) to final volume of 10 μl before macroarray hybridization.







TABLE 3







Amount of target species DNA, common co-inhabiting non-target fungal species DNA, and


host DNA used in 25 μl-PCR reactions for assessment of hybridization interference













Reaction
Target DNA
C. trifolii DNA
M. elongata DNA
T. virens DNA
Host DNA
Hybridization


number
amount (ng) a
amount (ng)
amount (ng)
amount (ng)
amount (ng)
signal observed b





1
10−4
0
0
0
1
yes


2
10−3
0
0
0
1
yes


3
10−2
0
0
0
1
yes


4
10−1
0
0
0
1
yes


5
10−2
 10−2
 10−2
 10−2
1
yes


6
10−1
 10−1
 10−1
 10−1
1
yes


7
1 
1
1
1
1
yes





a Target species used were Rhizoctonia solani isolate #98 or Pythium aphanidermatum isolate #99. The non-target fungal species used were Curvularia trifolii, Trichoderma virens, and Mortierella elongata while the host species was Poa annua.


b Yes implies that a hybridization signal was observed.






Array Validation

The macroarray was validated with DNA extracted from plant tissues or soils infested by the target species. DNA was extracted using UltraClean Soil DNA kit (MoBio Laboratories, Inc., Solana Beach, Calif., USA) or DNeasy Plant Mini kit (Qiagen Inc., Valencia, Calif., USA) depending on the material. Microscopic observation was also performed to check for presence of fungal or oomycete structures in the substrate.
Array Specificity
To assess the specificity of detection by the array, fungal and oomycete strains that differ from the target species by 1 to 3 bases of the probe sequence were tested for cross-reaction against the array (Table 4). DNA extraction, purification and hybridization were done as described above. Other fungal and oomycete species often found associated with turfgrasses were also included in the experiments to test the array's ability to discriminate non-target species.







TABLE 4





DNA sequence mismatches between target and closely related non-target


species at the ITS region where the probe was designed.






















Non-targeta



Target


Species



Speciesb
Probe
Probe sequence
with 1 nt mismatch
Mismatch position





Pa
Pa1
GGAGAGAGATGGCAGAATGTGAG
P. torulosum
GGAGAGAAATGGCAGAATGTGAG




(SEQ ID NO: 1)
(#122)
(SEQ ID NO: 61)


 



Pa2
GGGAGAGAGATGGCAGAATGTGAG
—
NA




(SEQ ID NO: 2)




 



Pa3
GAGGTGTACCTGAATTGTGTGAGG
—
NA




(SEQ ID NO: 3)




 


Rs
Rs2
CAGTGTTATGCTTGGTTCCACTC
—
NA




(SEQ ID NO: 16)




 



Rs3
TGTTGAAACTTAGTATTAGATGCGT
—
NA




(SEQ ID NO: 17)




 



Rs4
GAGTGGAACCAAGCATAACACTG
—
NA




(SEQ ID NO: 18)




 


Fs
Fs4
TCGCGTAGTAGCTAACACCTCGC
—
NA




(SEQ ID NO: 25)




 



Fs6
CCTGTGAACATACCTAAACGTTG
F. solani
CCTGTGAACATACCTAAAACGTTG




(SEQ ID NO: 26)
(#F19)
(SEQ ID NO: 67)


 



Fs13
TTATACAACTCATCAACCCTGTGA
F. solani
TTATTCAACTCATCAACCCTGTGA




(SEQ ID NO: 27)
(#F3)
(SEQ ID NO: 69)


 


Fo
Fo1
CGTTCCTCAAATTGATTGGCGGTC
—
NA




(SEQ ID NO: 40)




 



Fox2
GTTGGGACTCGCGTTAATTCG
—
NA




(SEQ ID NO: 41)






Species

Species



Target
with 2 nt

with 3 nt



Speciesb
mismatch
Mismatch position
mismatch
Mismatch positions





Pa
P. volutum
GGAGAGAAATGGCAGATGTGAG
—
NA



(#124p-1)
(SEQ ID NO: 62)




 



P. torulosum
AGGAGAGAAATGGCAGAATGTGAG
P. volutum
AGGAGAGAAATGGCAGATGTGAG



(#122)
(SEQ ID NO: 63)
(#124p-1)
(SEQ ID NO: 64)


 



P. volutum
GAGGTGTACCTGTCTTGTGTGAGG
—
NA



(#124p-1),
(SEQ ID NO: 65)





P. torulosum






(#122)





 


Rs
—
NA
—
NA



—
NA
—
NA



—
NA
—
NA


 


Fs
F. equiseti
TAGCGTGAGTAGCTAACACCTCGT
—
NA



(#S2)
(SEQ ID NO: 66)




 



F. equiseti
CCTGTGAACATACCTACGTTG
—
NA



(#S2)
(SEQ ID NO: 68)




 



F. oxysporum
TTATACAACTCATCAAACCCCTGT
—
NA



(#F2, #S4),
GA





F. equiseti
(SEQ ID NO: 70)





(#S2)





 


Fo
F. equiseti
CGTCCCTCAAATCGATTGGGGGTC
—
NA



(#S2)
(SEQ ID NO: 71)





—
NA
F. equiseti
GTTGGGACTCGCGGTAACCCG





(#S2)
(SEQ ID NO: 72)





aindicates no candidate species was available with the specified mismatch, NA = not applicable,


sequences that caused cross-hybridization are in boldface.


bPa = Phythium aphanidermatum, Rs = Rhizoctonia solani, Fs = Fusarium solani, Fo = Fusarium oxysporum













TABLE 5







Two-way ANOVA for hybridization results of monomers printed on a


membrane with and without other probe types, tested against four


isolates (N = 24).


ANOVA












Source of Variation
SS
df
MS
F
P-value















Monomer type
192.13
1
192.13
0.21
0.65


Isolate
5154.76
3
1718.25
1.92
0.17


Interaction (Monomer
257.45
3
85.82
0.09
0.96


type x Isolate)


Error
14349.09
16
896.82


Total
19953.44
23
















TABLE 6





Comparison of hybridization results between target and non-target species based on monomer, dimer, and dimer with poly-A spacer probes.







Probe target species/type/namec









Test
Pythium aphanidermatum
Rhizoctonia solani

























spec-

M
M
M
D
D
D
DA
DA
DA
M
M
M
D
D
D
DA
DA


iesa
ID #b
Pa1
Pa2
Pa3
Pa1R
Pa2R
Pa3R
Pa1R-A
Pa2R-A
Pa3R-A
Rs2
Rs3
Rs4
Rs2R
Rs3R
Rs4R
Rs2R-A
Rs3R-A





Ct
#185
 −d
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fe
#S2
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fo
#F2
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fo
#S4
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fs
#F3
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fs
#F19
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Me
#141
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Poa
NA
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pa
#60
 
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−


Pa
#99
 
+
+
+
+
+
+
+
+
−
−
−
−
−
−
−
−


Pr
#123p
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pt
#122
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pv
#124p-1
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Wc
#10
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Wc
#158
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Rs
#RH 20
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+


Rs
#98
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+
+
+


Tv
#126-L
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−










Probe target species/type/namec










Rhizoc-












tonia




Test
solani
Fusarium solani
Fusarium oxysporum























spe-
DA
M
M
M
D
D
D
DA
DA
DA
M
M
D
D
DA
DA


ciesa
Rs4R-A
Fs4
Fs6
Fs13
Fs4R
Fs6R
Fs13R
Fs4R-A
Fs6R-A
Fs13R-A
Fo1
Fox2
Fo1R
Fox2R
Fo1R-A
Fox2R-A





Ct
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Fe
−
−
−
−
−
−
−
−
−
−
−
−
⊕
−
⊕
−


Fo
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+


Fo
−
−
−
−
−
−
−
−
−
−
+
+
+
+
+
+


Fs
−
+
+
−
+
+
⊕
+
+
⊕
−
−
−
−
−
−


Fs
−
+
−
+
+
⊕
+
+
⊕
+
−
−
−
−
−
−


Me
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Poa
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pa
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pa
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pr
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pt
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Pv
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Wc
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Wc
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Rs
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Rs
+
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−


Tv
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−
−





aCt = Curvularia trifolii, Fe = Fusarium equiseti, Fo = Fusarium oxysporum, Fs = Fusarium solani, Me = Mortierella elongata, Poa = Poa annua, Pa = Pythium aphanidermatum, Pr = Pythium rostrum, Pt = Pythium torulosum, Pa = Pythium volutum, Wc = Waitea circinata var. circinata, Rs = Rhizoctonia solani, Tv = Trichoderma virens.


bCollection ID/accession number.


cA list of 33 probe names derived from 4 target species and 3 probe types, where M = monomer, D = dimer, and DA = dimer with 10 adenine (poly-A) spacer.


d− indicates no hybridization signal was observed, + indicates a hybridization signal was observed. All isolates were tested on three probes for each target in quadruplicate. False negatives ( ) were observed in M probe Pa1 for P. aphanidermatum test isolates (#60 and #99). False positives (⊕) between F. oxysporum probes (D and DA) and F. equiseti, and between F. solani probes (D and DA) and non-target F. solani strains.






Statistical Analysis

Every probe was spotted four times on the macroarray and measured individually, and the hybridization experiment was repeated at least once. Quantitative data were analyzed using a two-way ANOVA (isolate and probe type) with SAS (version 9.2) statistical software. A P value <0.05 was considered significant. Specifically, signal intensities of hybridization of monomers printed on a membrane with and without other probe types were analyzed using a 2×4 factorial design.
EXAMPLE 2
Additional Dimeric Oligonucleotide Probes
Additional dimeric probes that hybridize to target sequences that are specific to over 50 turf grass pathogens were designed as described in Example 1. The sequences for these probes, as well as the pathogenic target, are shown in Table 7. Positive and negative control probes are also included in this table. These probes may be evaluated and employed in macroarray analyses, as described above in Example 1.







TABLE 7







Additional dimeric oligonucleotide probes











Probe

Probe


Pathogen (Probe Target)
Name
Probe Sequence
Length





Brumeria graminis
Bg_2D
TGTAACTCTCCGCGTAGTAATATGTAACTCTCCGCGTAGTAATA
44




(SEQ ID NO: 73)




Bg_3D
GTTGACCCTCCACCCGTGTCGATTAGTTGACCCTCCACCCGTGTCGATTA
50




(SEQ ID NO: 74)




Bg_4D
AGCGTCCGTAACAACCTCTCAAGCCTAGCGTCCGTAACAACCTCTCAAGCCT
52




(SEQ ID NO: 75)




Bg_5D
GAGCGTCCGTAACAACCTCTCAAGCCTGAGCGTCCGTAACAACCTCTCAAGCCT
54




(SEQ ID NO: 76)



 


Bipolaris zeicola
Bz-4Da
TTTCGGAGCGCAGCACATATTTTGTTTCGGAGCGCAGCACATATTTTG
48




(SEQ ID NO: 77)




Bz-5Da
CTGGGAGACTCGCCTTAAAACGATTGCTGGGAGACTCGCCTTAAAACGATTG
52




(SEQ ID NO: 78)




Bz-6D
GCTTGGTGTTGGGCGTTTTTTGTCTCCGCTTGGTGTTGGGCGTTTTTTGTCTCC
54




(SEQ ID NO: 79)




Bz-7D
CATTTTTAACTTTTGACCTCGGATCATTTTTAACTTTTGACCTCGGAT
48




(SEQ ID NO: 80)



 


Colletotrichum cereale
Cc_2D
CTACCAGGGGACGTGGCGCCCGCCGCTACCAGGGGACGTGGCGCCCGCCG
50




(SEQ ID NO: 81)




Cc_3D
CCAGGGGACGTGGCGCCCGCCGGACCAGGGGACGTGGCGCCCGCCGGA
48




(SEQ ID NO: 82)




Cc_7D
ACGACGTTTCTTCTGAGTGGCACAACGACGTTTCTTCTGAGTGGCACA
48




(SEQ ID NO: 83)




Cc_9D
CTGAGTGGCACAAGCAAATAATTCTGAGTGGCACAAGCAAATAATT
46




(SEQ ID NO: 84)



 


Eudarluca caricis
Ec_2D
CAGAAACCGCTCTATACTCGGCAGAAACCGCTCTATACTCGG
42




(SEQ ID NO: 85)




Ec_3D
GCCTGATTCTCCCCATGTCTTGCCTGATTCTCCCCATGTCTT
42




(SEQ ID NO: 86)




Ec_6D
ACGATAGCCTGAAGCGCAGCACATACGATAGCCTGAAGCGCAGCACAT
48




(SEQ ID NO: 87)




Ec_7D
CAGCGTCAGTAACAAGTAATTCAGCGTCAGTAACAAGTAATT
42




(SEQ ID NO: 88)



 


Puccinia coronata
Pc_3D
AGAATAGAGTGCACTTGATTGTGGCTAGAATAGAGTGCACTTGATTGTGGCT
52




(SEQ ID NO: 89)




Pc_6D
TTATTAGGAGAGTTACATTACCCTTATTAGGAGAGTTACATTACCC
46




(SEQ ID NO: 90)




Pc_7D
CTTGGTTGCATGATTTGAAAGAGTCACTTGGTTGCATGATTTGAAAGAGTCA
48




(SEQ ID NO: 91)




Pc_9D
TTAAAAAGACTTGGTTGCATTTAAAAAGACTTGGTTGCAT
40




(SEQ ID NO: 92)



 


Puccinia persistens
Pp_t_1D
GCATTCTTTATTGAATGTTCACAGCATTCTTTATTGAATGTTCACA
48


var triticina

(SEQ ID NO: 93)




Pp_t_2D
CACTTCTTTGCATGATTTGAAAGACACTTCTTTGCATGATTTGAAAGA
48




(SEQ ID NO: 94)




Pp_t_4D
AATCTTACCCAAACTTTTAACACAATCTTACCCAAACTTTTAACAC
46




(SEQ ID NO: 95)




Pp_t_7D
GTTTAGTGGATGTTGAGTGTTGCTGTCGTTTAGTGGATGTTGAGTGTTGCTGTC
54




(SEQ ID NO: 96)



 


Puccinia striiformis
Ps_2D
ACGTAACTTCTTTATTGAATGTTGCACGTAACTTCTTTATTGAATGTTGC
50




(SEQ ID NO: 97)




Ps_7D
GTCACTTTTCTATAAGTTGGATGTCACTTTTCTATAAGTTGGAT
44




(SEQ ID NO: 98)




Ps_9D
CATCTTATTTAAGGGAGACTCCATCTTATTTAAGGGAGACTC
44




(SEQ ID NO: 99)




Ps_10D
GAGACTCCTAAAAACCCAATGAGACTCCTAAAAACCCAAT
40




(SEQ ID NO: 100)



 


Puccinia graminis
Pg_1D
ACTTTTAAAAACTTGGTTGCATGAACTTTTAAAAACTTGGTTTGCATGA
48




(SEQ ID NO: 101)




Pg_5D
TTAGTGGATGTTGAGTGTTGCTGTACCTTAGTGGATGTTGAGTGTTGCTGTACC
54




(SEQ ID NO: 102)




Pg_7D
CACTTGCCATCTTGTTTGTTACACTTGCCATCTTGTTTGTTA
42




(SEQ ID NO: 103)




Pg_8D
GAGTATACGTAACATTCTTAATTGAGAGTATACGTAACATTCTTAATTGA
50




(SEQ ID NO: 104)



 


Puccinia graminis f.
Pg_T1d
GCCATCTTTTTTGTAACAAGAGACGCCATCTTTTTTGTAACAAGAGAC
48


sp. Tritici

(SEQ ID NO: 105)




Pg_T2d
CCCAATATCTATTTTTTTTAAGACCTCCCAATATCTATTTTTTTTAAGACCT
52




(SEQ ID NO: 106)




Pg_T4d
AACAAGAGACTCCTAAAACCCAAACAAGAGACTCCTAAAACCCA
44




(SEQ ID NO: 107)




Pg_T3d
TGGGTTTTAGGAGTCTCTTGTTTGGGTTTTAGGAGTCTCTTGTT
44




(SEQ ID NO: 108)



 


Pythium volutum
Pv_2D
GTTCTGTGCCTTCTCTTGGGAGGTTCTGTGCCTTCTCTTGGGAG
44




(SEQ ID NO: 109)




Pv_3D
GAAGGTTGGCTGCAAATGTAGTGAAGGTTGGCTGCAAATGTAGT
48




(SEQ ID NO: 110)




Pv_4D
CTGTATGCGCGGTCTTCCGATGTACTGTATGCGCGGTCTTCCGATGTA
48




(SEQ ID NO: 111)




Pv_10D
CTTGTGTTTGAGAGAAGTGCTGACCCTTGTGTTTGAGAGAAGTGCTGACC
50




(SEQ ID NO: 112)



 


Pythium torulosum/
Pt_1D
GCGGTTTTGCCGATGTACTTTTAAACGCGGTTTTGCCGATGTACTTTTAAAC
52




(SEQ ID NO: 113)




Pt_2D
GTACCTGTCTTGTGTGAGGCAACGGTACCTGTCTTGTGTGAGGCAACG
48




(SEQ ID NO: 114)




Pt_3D
TGTCTTGTGTGAGGCAACGGTCTGTGTCTTGTGTGAGGCAACGGTCTG
48




(SEQ ID NO: 115)




Pt_4D
GTACCTGTCTTGTGTGAGGCAACGGGTACCTGTCTTGTGTGAGGCAACGG
50




(SEQ ID NO: 116)



 


Pythium arrhenomanes
Parr_1D
TGTAATTTTGTTTTGTGCCTTCTTTCTGTAATTTTGTTTTGTGCCTTCTTTC
52




(SEQ ID NO: 117)




Parr_2D
GAAAGAAGGCACAAAACAAAATTACAGAAAGAAGGCACAAAACAAAATTACA
52




(SEQ ID NO: 118)



 


Pythium deliense
Pd_1D
TACCTGATTTGTGTGAGGCAATGGTTACCTGATTTGTGTGAGGCAATGGT
50




(SEQ ID NO: 119)




Pd_2D
TCATGTTCTGTGCTCTCTCTCGGGATCATGTTCTGTGCTCTCTCTCGGGA
50




(SEQ ID NO: 120)




Pd_3D
CGTTGACTCCCTTTTCGGAGGAGAACGTTGACTCCCTTTTCGGAGGAGAA
50




(SEQ ID NO: 121)




Pd_4D
GCTTAATTGTGGTCTGCCGATGTATTTGCTTAATTGTGGTCTGCCGATGTATTT
54




(SEQ ID NO: 122)



 


Pythium rostratifingens
Pr_2D
GAGTCGGCTAAACGAAGGTCGGGGAGTCGGCTAAACGAAGGTCGGG
46




(SEQ ID NO: 123)




Pr_5D
GACTCCGGTTTTTCTATTGCGTTGCTGACTCCGGTTTTTCTATTGCGTTGCT
52




(SEQ ID NO: 124)




Pr_6D
TTGGAGAAGGAGCAGAGGTGAAGTTGGAGAAGGAGCAGAGGTGAAG
44




(SEQ ID NO: 125)




Pr_10D
CTCCAGAGCACGCTACCGAGGTCTCCAGAGCACGCTACCGAGGT
44




(SEQ ID NO: 126)



 


Pythium rostratum
Prm_1D
GGACTGATGTGCGCTTGTCGCATGTGGACTGATGTGCGCTTGTCGCATGT
50




(SEQ ID NO: 127)




Prm_6D
TTAAACCATACCATAAGTACTGATTTTAAACCATACCATAAGTACTGATT
50




(SEQ ID NO: 128)




Prm_9D
TCTCCGCTGAGAGTTTGTGTGTGTCTCCGCTGAGAGTTTGTGTGTG
54




(SEQ ID NO: 129)




Prm_10D
CTCTCCGCTGAGAGTTTGTGTGTGCTCTCCGCTGAGAGTTTGTGTGTG
42




(SEQ ID NO: 130)



 


Pyrhium aphanidermatum
Pa_1D
GCTGCTCTTGGACGCCCTGTTTTCGCTGCTCTTGGACGCCCTGTTTTC
52




(SEQ ID NO: 131)




Pa_2D
GCTGCTCTTGGACGCCCTGTTGCTGCTCTTGGACGCCCTGTT
42




(SEQ ID NO: 132)




Pa_4D
GACTGTTTGCAATTTATTGTGAGACTGTTTGCAATTTATTGTGA
44




(SEQ ID NO: 133)




Pa_5D
GAAAGTTTATGGTTTTAATCTAGAAAGTTTATGGTTTTAATCTA
44




(SEQ ID NO: 134)



 


Pythium myriotylum
Pm_1D
GATTAGAGATGGCAGAATGTGAGGTGGATTAGAGATGGCAGAATGTGAGGTG
52




(SEQ ID NO: 135)




Pm_2D
GCTCTGCGCGAGTGGGCGACTTCGGTGCTCTGCGCGAGTGGGCGACTTCGGT
52




(SEQ ID NO: 136)




Pm_4D
CCTGTCTTGTGTGGGGCAATGGTCTGCCTGTCTTGTGTGGGGCAATGGTCTG
52




(SEQ ID NO: 137)




Pm_5D
CCTGTCTTGTGTGGGGCAATGGTCCTGTCTTGTGTGGGGCAATGGT
46




(SEQ ID NO: 138)



 


Pythium arrhenomanes
Par_3D
GGTTGTCCGCAAGTGTAGTTAATTCGGTTGTCCGCAAGTGTAGTTAATTC
50




(SEQ ID NO: 139)




Par_5D
AGATGGCAGATGTGAGGTGTCTAGATGGCAGATGTGAGGTGTCT
44




(SEQ ID NO: 140)




Par_6D
GTGTCTGAGAGAAGTGTGACCTGTGTCTGAGAGAAGTGTGACCT
44




(SEQ ID NO: 141)




Par_7D
AGTGGTTATTGCTCTTGGACGCAGTGGTTATTGCTCTTGGACGC
44




(SEQ ID NO: 142)



 


Rhizotonia solani
Rs_2D
AACGAATGTAATTGATGTAACGAACGAATGTAATTGATGTAACG
44




(SEQ ID NO: 143)




Rs_4D
CTGGATCTCAGTGTTATGCTTGGCTGGATCTCAGTGTTATGCTTGG
46




(SEQ ID NO: 144)




Rs_5D
ACCGCTTCTAATAGTCCATTGACACCGCTTCTAATAGTCCATTGAC
46




(SEQ ID NO: 145)




Rs_9D
GTAACGCATCTAATACTAAGTTTGTAACGCATCTAATACTAAGTTT
46




(SEQ ID NO: 146)



 


Ceratobasidium cereale
Cr_4D
CCGTCCAATACATAAAATCTTACCGTCCAATACATAAAATCTTA
44




(SEQ ID NO: 147)




Cr_6D
TAATCAGAATGTAATCGATGTAAACGTAATCAGAATGTAATCGATGTAAACG
52




(SEQ ID NO: 148)




Cr_7D
GTAAACGCATCTATAAACTAAGGTAAACGCATCTATAAACTAAG
44




(SEQ ID NO: 149)




Cr_8D
GGCTTTTGTTTTGGATTTGGAGGTGGCTTTTGTTTTGGATTTGGAGGT
48




(SEQ ID NO: 150)



 


Waitea circinata
Wc_1D
GTCCCTGTAGACTCTGCTTCAGTCCCTGTAGACTCTGCTTCA
42




(SEQ ID NO: 151)




Wc_2D
CTAGTGTTTCTAGTATGTGCACTAGTGTTTCTAGTATGTGCA
42




(SEQ ID NO: 152)




Wc_3D
GTAATAGATCTATGTGGATACGGTAATAGATCTATGTGGATACG
44




(SEQ ID NO: 153)




Wc_3D
TGAAGCAGAGTCTACAGGGACTGAAGCAGAGTCTACAGGGAC
42




(SEQ ID NO: 154)



 


Rhizoctonia zeae
Wcz_1D
CTTCTGTAATAGATCTATGTGGATACGCTTCTGTAATAGATCTATGTGGATACG
54




(SEQ ID NO: 155)




Wcz_2D
CATGAATCTCTCAAATACAATGATTTCATGAATCTCTCAAATACAATGATTT
52




(SEQ ID NO: 156)




Wcz_3D
CATGAATCTCTCAAATACAATGACATGAATCTCTCAAATACAATGA
46




(SEQ ID NO: 157)




Wcz_4D
CCTTCTGTAATAGATCTATGTGCCTTCTGTAATAGATCTATGTG
44




(SEQ ID NO: 158)



 


Waitea circinata
Wcc_4D
TTATACACACACAATAGTCATTGTTATACACACACAATAGTCATTG
46


var. circinata

(SEQ ID NO: 159)




Wcc_8D
CCTGTGCACCTTTTGTAGTATTACCCTGTGCACCTTTTGTAGTATTAC
48




(SEQ ID NO: 160)




Wcc_11D
CAAATGTATTAGCTGGGGTTTATATAGCAAATGTATTAGCTGGGGTTTATATAG
54




(SEQ ID NO: 161)




Wcc_12D
TGGAAGCTGTTGGCGCAAGTCGATGGAAGCTGTTGGCGCAAGTCGA
50




(SEQ ID NO: 162)



 


Rhizotonia oryzae
Wco_1D
TATTTTGAATCATTATTATTTGGACTATTTTGAATCATTATTATTTGGAC
50




(SEQ ID NO: 163)




Wco_2D
CTTGGAAGTTTGTCGGCGCAAGTCTTGGAAGTTTGTCGGCGCAAGT
46




(SEQ ID NO: 164)




Wco_3D
TGAGTGTCATGAATCTCTCAAATATGAGTGTCATGAATCTCTCAAATA
48




(SEQ ID NO: 165)




Wco_4D
ATTTGGACTTGGAAGTTTGTCGGCATTTGGACTTGGAAGTTTGTCGGC
48




(SEQ ID NO: 166)




Wco_6D
TTGGAAGTTTGTCGGCGCAAGTTTGGAAGTTTGTCGGCGCAAGT
46




(SEQ ID NO: 167)



 


Sclerotinia homoeocarpa
Sh_1D
TCCAACCCTTGTGTATCTCTACCATCCAACCCTTGTGTATCTCTACCA
48




(SEQ ID NO: 168)




Sh_2D
CCTTGTGTATCTCTACCATGTTCCTTGTGTATCTCTACCATGTT
44




(SEQ ID NO: 169)




Sh_4D
ACAGCCTCAGCGCCCTCCGGGGCCACAGCCTCAGCGCCCTCCGGGGCC
48




(SEQ ID NO: 170)




Sh_5D
AGGAAAATCACAACTCTGAATTGAGGAAAATCACAACTCTGAATTG
46




(SEQ ID NO: 171)



 


Typhula incarnata
Ta_2D
ATGGGGTTCTGCTTCTAATCGTCATGGGGTTCTGCTTCTAATCGTC
46




(SEQ ID NO: 172)




Ta_3D
CTCTTTGTGGTGCCAGACTATGCTCTTTGTGGTGCCAGACTATG
44




(SEQ ID NO: 173)




Ta_5D
GTGATAATTATCTACGCTGTGGTTGTGATAATTATCTACGCTGTGGTT
48




(SEQ ID NO: 174)




Ta_6D
GCTGCGAATTTAACTATGGGGCTGCGAATTTAACTATGGG
40




(SEQ ID NO: 175)



 


Typhula ishikariensis
Ti_2D
CTACGCTGTTGGTCTTGTGAACTACGCTGTTGGTCTTGTGAA
42




(SEQ ID NO: 176)




Ti_3D
GGTCTTGTGAAGCACTTTATTGTGGTCTTGTGAAGCACTTTATTGT
46




(SEQ ID NO: 177)




Ti_4D
GTTACGAGGTTCTGCTTCTAATCGTTACGAGGTTCTGCTTCTAATC
46




(SEQ ID NO: 178)




Ti_5D
TTCTAATCGTCCTTTACTGCTTCTAATCGTCCTTTACTGC
40




(SEQ ID NO: 179)



 


Gaeumannomyces graminis
Gg_1D
CTGTTGCTTCGGCGGACGATGGCTGTTGCTTCGGCGGACGATGG
44




(SEQ ID NO: 180)




Gg_2D
GACGCCGCCGGAGGTTACAAACCGACGCCGCCGGAGGTTACAAACC
46




(SEQ ID NO: 181)




Gg_3D
GGACGCCGCCGGAGGTTACAGGACGCCGCCGGAGGTTACA
40




(SEQ ID NO: 182)




Gg_4D
CCGCCCGGCGGTCGGGGCCCCCACCGCCCGGCGGTCGGGGCCCCCA
42




(SEQ ID NO: 183)



 


Magnaporthe grisea
Mg_1D
CAACCCTCAAGCCCCGGCTTGGTCAACCCTCAAGCCCCGGCTTGGT
46




(SEQ ID NO: 184)




Mg_2D
GCATCTCTGAGCCTAAAAGACAAGCATCTCTGAGCCTAAAAGACAA
46




(SEQ ID NO: 185)






46



Mg_5D
GAACCCTCGCTCGGCCCGTCACCGAACCCTCGCTCGGCCCGTCACC
46




(SEQ ID NO: 186)



 


Magnaporthe oryzae
Mo_1D
GCCTCGGCTTGGTGTTGGGGCGCCTCGGCTTGGTGTTGGGGC
42




(SEQ ID NO: 187)




Mo_5D
CACGCCCGCCGGAGGTTCAAAACTCACGCCCGCCGGAGGTTCAAAACT
48




(SEQ ID NO: 188)




Mo_6D
CGCCGGAGGTTCAAAACTCTTATTCGCCGGAGGTTCAAAACTCTTATT
40




(SEQ ID NO: 189)




Mo_10D
GTGCTCCAGCCGCTAAACCCCCAATTCGTGCTCCAGCCGCTAAACCCCCAATTC
50




(SEQ ID NO: 190)



 


Magnaporyhe poae
Mp_1D
CGCCGCCGGAGGTTCAAAACCCGCCGCCGGAGGTTCAAAACC
42




(SEQ ID NO: 191)




Mp_2D
CCGCCGGAGGTTCAAAACCCTCCGCCGGAGGTTCAAAACCCT
42




(SEQ ID NO: 192)




Mp_3D
AACGCGCCCTCGCTCGGCGGCAACGCGCCCTCGCTCGGCGGC
42




(SEQ ID NO: 193)






42


 


Gaeumannomyces
Gi_1D
GCTCCGAGCGCAGTAGCACGCGCTCCGAGCGCAGTAGCACGC
42


incrustans

(SEQ ID NO: 194)




Gi_2D
GGTTGGCGCCGGTGCCCAGATGGGTTGGCGCCGGTGCCCAGATG
44




(SEQ ID NO: 195)




Gi_3D
GTCGCCGCCGGAGGTTCGAAACCGTCGCCGCCGGAGGTTCGAAACC
46




(SEQ ID NO: 196)




Gi_4D
CCGCCGGAGGTTCGAAACCCTCCGCCGGAGGTTCGAAACCCT
42




(SEQ ID NO: 197)



 


Magnaporthe rhizophila
Mr_1D
TTCGAAACCCTGAATTCTAGTGTTCGAAACCCTGAATTCTAGTG
44




(SEQ ID NO: 198)




Mr_2D
GAGGTCGCCGCCGGAGGTTCGAAGAGGTCGCCGCCGGAGGTTCGAA
46




(SEQ ID NO: 200)




Mr_3D
GCCTGGAGGTCGCCGCCGGAGGTTCGCCTGGAGGTCGCCGCCGGAGGTTC
50




(SEQ ID NO: 201)




Mr_4D
CCAGATGGGCCTGGAGGTCGCCGCCCAGATGGGCCTGGAGGTCGCCGC
48




(SEQ ID NO: 202)



 


Magnaporthe salvinii
Ms_1D
AAGTACATCGGCGGACCCGCTGGGAAGTACATCGGCGGACCCGCTGGG
48




(SEQ ID NO: 203)




Ms_2D
GGCGGACCCGCTGGGGCCCTGAGGGCGGACCCGCTGGGGCCCTGAG
46




(SEQ ID NO: 204)




Ms_3D
CGCCTCGCTCGGTGGATCCCCGGAGGCGCCTCGCTCGGTGGATCCCCGGAGG
52




(SEQ ID NO: 205)




Ms_4D
CCGGAGGGCATTCCAGCCGCTAAACCGGAGGGCATTCCAGCCGCTAAA
48




(SEQ ID NO: 206)



 


Microdochium bolleyi
Mb_1D
CTGGAAACAGTGCTGCCACCGGTGGACTGGAAACAGTGCTGCCACCGGTGGA
46




(SEQ ID NO: 207)




Mb_4D
AAGCCGGCCAGACGACAGCCATAAAAGCCGGCCAGACGACAGCCATAA
48




(SEQ ID NO: 208)




Mb_5D
GCCAGACGACAGCCATAAACCGCCAGACGACAGCCATAAACC
42




(SEQ ID NO: 209)




Mb_6D
CTGGAAACAGTGCTGCCACCGGTCTGGAAACAGTGCTGCCACCGGT
46




(SEQ ID NO: 210)



 


Microdochium nivale
Mn_2D
GGTGGATGGTGCTGTCTCTCGGGTGGATGGTGCTGTCTCTCG
42




(SEQ ID NO: 211)




Mn_4D
TGGACTACCTAAACTCTGTTATGGACTACCTAAACTCTGTTA
42




(SEQ ID NO: 212)




Mn_5D
GTCAATCTGAATCAAACTAAGGTCAATCTGAATCAAACTAAG
42




(SEQ ID NO: 213)




Mn_9D
CGGAGTCGGTTCGTGCTCTGACGGAGTCGGTTCGTGCTCTGA
42




(SEQ ID NO: 214)



 


Gleocercospora sorghi
Gs_1D
CTCGGTGGTTAGTACTCTCTCTCGCTCGGTGGTTAGTACTCTCTCTCG
48




(SEQ ID NO: 215)




Gs_2D
CGGTGGTTAGTGCTCTCTCTCGGCGGTGGTTAGTGCTCTCTCTCGG
46




(SEQ ID NO: 216)




Gs_4D
TCTCTCTCGGGAGGGTGCTGCCTCTCTCTCGGGAGGGTGCTGCC
44




(SEQ ID NO: 217)




Gs_6D
GTAATTACTTATCTCGCTTCTTGTAATTACTTATCTCGCTTCTT
44




(SEQ ID NO: 218)



 


Laetisaria fuciformis
Lf_1D
CCTTTGGGTGTCCGAGTTGTATTCCTTTGGGTGTCCGAGTTGTATT
46




(SEQ ID NO: 219)




Lf_2D
TTTCCGCGCTGGACTGTGTAAATTTCCGCGCTGGACTGTGTAAA
44




(SEQ ID NO: 220)




Lf_3D
CGCGTTGTATGAGACTCAGCCTCGCGTTGTATGAGACTCAGCCT
44




(SEQ ID NO: 221)




Lf_5D
GGCATCCTTTGGGTGTCCGAGTTGGGCATCCTTTGGGTGTCCGAGTTG
48




(SEQ ID NO: 222)



 


Leptosphaeria korrae
Lk_1D
TAAAGCAATTGGCAGCCTATATCTTAAAGCAATTGGCAGCCTATATCT
48




(SEQ ID NO: 223)




Lk_2D
AGCACAAACTGCATGGGCGGAGCACAAACTGCATGGGCGG
40




(SEQ ID NO: 224)




Lk_3D
CCCATTGAACCTATTTATTTTCCCATTGAACCTATTTATTTT
42




(SEQ ID NO: 225)




Lk_6D
AGCAATTGGCAGCCTATATCTGGAGCAATTGGCAGCCTATATCTGG
46




(SEQ ID NO: 226)



 


Ophiosphaerella
Oh_1D
TCTTACTGCCAGTTATATAGGCACTCTTACTGCCAGTTATATAGGCAC
48


herpotricha

(SEQ ID NO: 227)




Oh_2D
GTGTAGAACAAACTACGCAGACGTGTAGAACAAACTACGCAGAC
44




(SEQ ID NO: 228)




Oh_3D
CCAATAAGCCTTTTTATCACCCAATAAGCCTTTTTATCAC
40




(SEQ ID NO: 229)




Oh_4D
TCTACTCCACTGCGTTTGGACTCGTCTACTCCACTGCGTTTGGACTCG
48




(SEQ ID NO: 230)



 


Ophiosphaerella
Oa_1D
AGAACATAGGCCCCAAGCTGTAGCAGAACATAGGCCCCAAGCTGTAGC
48


agrostis

(SEQ ID NO: 231)




Oa_4D
AAGGCCTCTTCTATTACCCTTGTAAGGCCTCTTCTATTACCCTTGT
46




(SEQ ID NO: 232)




Oa_5D
ATCATTACATTAGAACATAGGCCATCATTACATTAGAACATAGGCC
46




(SEQ ID NO: 233)




Oa_11D
GGTGTTTTGTCCTCTCCATTGCGGGTGTTTTGTCCTCTCCATTGCG
46




(SEQ ID NO: 234)



 


Limonomyces roseipellis
Lr_1D
ATATCAATAACACAAACTAACAAGATATCAATAACACAAACTAACAAG
48




(SEQ ID NO: 235)




Lr_2D
CTGGCATCCTCCGGGTGTCCGAGTCTGGCATCCTCCGGGTGTCCGAGT
48




(SEQ ID NO: 236)




Lr_3D
CTTGTTAGTTTGTGTTATTGATATCTTGTTAGTTTGTGTTATTGATAT
48




(SEQ ID NO: 237)




Lr_4D
ACTCGGACACCCGGAGGATGCCAGACTCGGACACCCGGAGGATGCCAG
48




(SEQ ID NO: 238)



 


Acidovorax avenae
Aa_1D
ATAAAGGGAGGTCATGACGGTATAAAGGGAGGTCATGACGGT
42




(SEQ ID NO: 239)




Aa_2D
CTAATAAAGGGAGGTCATGACGCTAATAAAGGGAGGTCATGACG
44




(SEQ ID NO: 240)




Aa_3D
CGTCATGACCTCCCTTTATTAGCGTCATGACCTCCCTTTATTAG
44




(SEQ ID NO: 241)




Aa_4D
ACCGTCATGACCTCCCTTTATACCGTCATGACCTCCCTTTAT
42




(SEQ ID NO: 242)



 


Xanthomonas translucens
Xt-p_1D
AGTGAAATGCGTAAGATCGGGAGGAAGTGAAATGCGTAAGATCGGGAGGA
50


pv. poae

(SEQ ID NO: 243)




Xt-p_2D
CACGGAACTTTCCAGAGATGGATTGCACGGAACTTTCCAGAGATGGATTG
50




(SEQ ID NO: 244)




Xt-p_3D
CACAGTGGTAGCAATACCATGGGTGCACAGTGGTAGCAATACCATGGGTG
50




(SEQ ID NO: 245)




Xt-p_4D
CACAGTGGTAGCAATACCATGCACAGTGGTAGCAATACCATG
42




(SEQ ID NO: 246)



 


Curvularia trifolii
ct_1
GGGCGTCTTGTCTTTTGGCTCTGGGCGTCTTGTCTTTTGGCTCT
44




(SEQ ID NO: 247)




ct_2
GGCTCTTTGCCCAAAGACTCGGCTCTTTGCCCAAAGACTC
40




(SEQ ID NO: 248)




ct_3
CGCCAGGACCACACCATAAACCTCGCCAGGACCACACCATAAACCT
46




(SEQ ID NO: 249)




ct_4
GCCGCCAGGACCACACCATAAGCCGCCAGGACCACACCATAA
42




(SEQ ID NO: 250)



 


Trichoderma virens
tv_1
CGTTACCAAACTGTTGCCTCGGCCGTTACCAAACTGTTGCCTCGGC
46




(SEQ ID NO: 251)




tv_4
CAACCCTCGAACCCCTCCGGGCAACCCTCGAACCCCTCCGGG
42




(SEQ ID NO: 252)




tv_5
GTATTCTGGCGGGCATGCCTGTCCGTATTCTGGCGGGCATGCCTGTCC
48




(SEQ ID NO: 253)



 


Urocystis agropyri
Ua_1
GATCTGTATCCGCCCCCGACCCGATCTGTATCCGCCCCCGACCC
44




(SEQ ID NO: 254)




Ua_3
GTATCCGCCCCCGACCCTTCGATCGTATCCGCCCCCGACCCTTCGATC
48




(SEQ ID NO: 255)




Ua_4
GAGGGTAGCGCCGTTTCATGGTCGGAGGGTAGCGCCGTTTCATGGTCG
48




(SEQ ID NO: 256)




Ua_5
CTAATCTAGGAGTGGCATCGAACTAATCTAGGAGTGGCATCGAA
44




(SEQ ID NO: 257)



 


Ustilago striiformis
Us_1
CGCCCATATCGAGTTTTGCCTCGGCGCCCATATCGAGTTTTGCCTCGG
48




(SEQ ID NO: 258)




Us_2
TTACAATGAAATCGACTGGTAATGCTTACAATGAAATCGACTGGTAATGC
50




(SEQ ID NO: 259)




Us_3
ACAATGAAATCGACTGGTAATGCACAATGAAATCGACTGGTAATGC
46




(SEQ ID NO: 260)



 


Lycoperdon spp
Ly_1
GCACACTTGTCTTGACTTTATTCGCACACTTGTCTTGACTTTATTC
46




(SEQ ID NO: 261)




Ly_4
GGAGCATGTGCACACTTGTCTTGGAGCATGTGCACACTTGTCTT
44




(SEQ ID NO: 262)




Ly_5
CGAGTTGTGATGGGGCTTGGATCCGAGTTGTGATGGGGCTTGGATC
46




(SEQ ID NO: 263)



 


Bovista
Bov_1
TCCGGATGTGAGGAATTGCTGAGTTCCGGATGTGAGGAATTGCTGAGT
48




(SEQ ID NO: 264)




Bov_2
TACCTCTCCTTCAAGTACTATGTTACCTCTCCTTCAAGTACTATGT
46




(SEQ ID NO: 265)




Bov_4
ATTAAATTCTCAACCCCTCTAGCTTTATTAAATTCTCAACCCCTCTAGCTTT
52




(SEQ ID NO: 266)




Bov_6
AAATTCTCAACCCCTCTAGCTTAAATTCTCAACCCCTCTAGCTT
44




(SEQ ID NO: 267)



 


Agaricus
Ag_1
TGGACTTCATTTTCATCCACCTGTGGACTTCATTTTCATCCACCTG
46




(SEQ ID NO: 268)




Ag_6
TCTTTTTCCTGTTAGAGTCTATGTTCTTTTTCCTGTTAGAGTCTATGT
48




(SEQ ID NO: 269)




Ag_7
TTGTAGTCTTTTTCAGGTATTGTTGTAGTCTTTTTCAGGTATTG
44




(SEQ ID NO: 270)




Ag_8
GTTGTAAAGGAGAGCTTGGATTGTGTTGTAAAGGAGAGCTTGGATTGT
48




(SEQ ID NO: 271)



 


Marasmius
Mar_1
TTGGTATTCCGAGAGGCATGCCTGTTGGTATTCCGAGAGGCATGCCTG
48




(SEQ ID NO: 272)




Mar_2
TTGCGCCTCTTGGTATTCCGAGAGGTTGCGCCTCTTGGTATTCCGAGAGG
50




(SEQ ID NO: 273)



 


Lepiota
Le_1
CATGTAGTATGTTGCCAGAATGCATGTAGTATGTTGCCAGAATG
44




(SEQ ID NO: 274)




Le_2
ACCATGTAGTATGTTGCCAGAATGACCATGTAGTATGTTGCCAGAATG
48




(SEQ ID NO: 275)




Le_3
TATCACAAACCATGTAGTATGTTTATCACAAACCATGTAGTATGTT
46




(SEQ ID NO: 276)



 


Athelia rolfsii
At_2
ACATAGAACGATCTCATATTGAAACATAGAACGATCTCATATTGAA
46




(SEQ ID NO: 277)




At_3
ACTCTTATTGTATGTTACATAGAACACTCTTATTGTATGTTACATAGAAC
50




(SEQ ID NO: 278)




At_6
AGAGTCATTAAATTCTCAACCTTAGAGTCATTAAATTCTCAACCTT
46




(SEQ ID NO: 279)




At_7
CAAGGCTTGGATGTGAGAGTTGCTCAAGGCTTGGATGTGAGAGTTGCT
48




(SEQ ID NO: 280)



 


Gibberella zeae
Gz_3
AAGGGACGGCCCGCCGCAGGAACCCAAGGGACGGCCCGCCGCAGGAACCC
50




(SEQ ID NO: 281)




Gz_5
CTGCACTCCCCAAATACATTGGCGCTGCACTCCCCAAATACATTGGCG
48




(SEQ ID NO: 282)




Gz_6
GCTGCACTCCCCAAATACATTGGCGCTGCACTCCCCAAATACATTGGC
48




(SEQ ID NO: 283)



 


Fusarium oxysporum
Foxy_3
GGACTCGCGTTAATTCGCGTTCCGGACTCGCGTTAATTCGCGTTCC
46




(SEQ ID NO: 284)




Foxy_4
CGCGTTCCTCAAATTGATTGGCGGTCGCGTTCCTCAAATTGATTGGCGGT
50




(SEQ ID NO: 285)




Fo1R
CGTTCCTCAAATTGATTGGCGGTCCGTTCCTCAAATTGATTGGCGGTC
48




(SEQ ID NO: 42)




Fox2R
GTTGGGACTCGCGTTAATTCGGTTGGGACTCGCGTTAATTCG
42




(SEQ ID NO: 43)



 


Fusarium spp
Fus_1
TGTTGCCTCGGCGGATCAGCCCGCTGTTGCCTCGGCGGATCAGCCCGC
48




(SEQ ID NO: 286)




Fus_2
AAATAAATCAAAACTTTCAACAAAAATAAATCAAAACTTTCAACAA
46




(SEQ ID NO: 287)



 


Poa annua (RBCL)
Poa_R_1
CCTCAGCCTGGAGTTCCCCCGGACCTCAGCCTGGAGTTCCCCCGGA
46




(SEQ ID NO: 288)




Poa_R_2
ACATTGAGCCTGTTGCTGGGGAAGATACATTGAGCCTGTTGCTGGGGAAGAT
52




(SEQ ID NO: 289)




Poa_R_4
CTCAGCCTGGAGTTCCCCCGGACTCAGCCTGGAGTTCCCCCGGA
44




(SEQ ID NO: 290)



 


Lolium perenne (RBCL)
Lp_R_1
CATATCGAGCCTGTTGCTGGGGAAGACATATCGAGCCTGTTGCTGGGGAAGA
52




(SEQ ID NO: 291)




Lp_R_3
TATCGAGCCTGTTGCTGGGGAAGACATATCGAGCCTGTTGCTGGGGAAGACA
52




(SEQ ID NO: 292)



 


Agrostis stolonifera
As_R_1
AGTCCTCAACCTGGGGTTCCGCCGAGTCCTCAACCTGGGGTTCCGCCG
48


(RBCL)

(SEQ ID NO: 293)




As_R_2
AGTCCTCAACCTGGGGTTCCGCCGGAAGTCCTCAACCTGGGGTTCCGCCGGA
52




(SEQ ID NO: 294)



 


Poa annua (matK)
Poa_m_3
CGAGTAAGATGGAACATTTTGGCGAGTAAGATGGAACATTTTGG
44




(SEQ ID NO: 295)



 


Poa pratensis (matK)
Pop_m_1
TGCCAAAATTCGATACCATAGTTCTGCCAAAATTCGATACCATAGTTC
48




(SEQ ID NO: 296)




Pop_m_2
GAATGCCAAAATTCGATACCATAGTGAATGCCAAAATTCGATACCATAGT
50




(SEQ ID NO: 297)



 


Agrostis stolonifera
As_m_1
CTATCCATTTTGAAATCTTGGTGCACTATCCATTTTGAAATCTTGGTGCA
50


(matK)

(SEQ ID NO: 298)




As_m_2
GCAACTCCTTCAATACCGTATCAAGCAACTCCTTCAATACCGTATCAA
48




(SEQ ID NO: 299)




As_m_4
ATTATCTTCTGGAACTTTTCTGGAATTATCTTCTGGAACTTTTCTGGA
48




(SEQ ID NO: 300)




As_m_5
TGCAACTCCTTCAATACCGTATCAATGCAACTCCTTCAATACCGTATCAA
50




(SEQ ID NO: 301)



 


Positive control
ITS2R
GCTGCGTTCTTCATCGATGCGCTGCGTTCTTCATCGATGC
40




(SEQ ID NO: 48)



Negative controlb
ITS2_2_IR
GCTGCGTTGATCATCGATGCGCTGCGTTGATCATCGATGC
40




(SEQ ID NO: 57)



 


Positive control
ITS4R
TCCTCCGCTTATTGATATGCTCCTCCGCTTATTGATATGC
40




(SEQ ID NO: 49)



Negative control
ITS4_2_1R
TCCTCCGTTTATTGATTTGCTCCTCCGTTTATTGATTTGC
40




(SEQ ID NO: 302)



Negative control
ITS4_2_2R
TCCTCCGTTTATTGAAATGCTCCTCCGTTTATTGAAATGC
40




(SEQ ID NO: 303)



Negative control
ITS4_2_3R
TCCTCCGTTTATTGGTATGCTCCTCCGTTTATTGGTATGC
40




(SEQ ID NO: 304)



 


Positive control
matK-390F
CGATCTATTCATTCAATATTTCCGATCTATTCATTCAATATTTC
44




(SEQ ID NO: 305)



Positive control
matK-1326R
TCTAGCACACGAAAGTCGAAGTTCTAGCACACGAAAGTCGAAGT
44




(SEQ ID NO: 306)



 


Positive control
rbcLaF
ATGTCACCACAAACAGAGACTAAAGCATGTCACCACAAACAGAGACTAAAGC
52




(SEQ ID NO: 307)



Positive control
rbcL-1F
ATGTCACCACAAACAGAAACATGTCACCACAAACAGAAAC
40




(SEQ ID NO: 308)





Negative control probe sequences contain nucleotide mismatches as compared to the corresponding


positive control probe sequence; these nucleotides are shown in bold.






All documents cited herein are incorporated by reference. While certain embodiments of invention are described, and many details have been set forth for purposes of illustration, certain of the details can be varied without departing from the basic principles of the invention.
The use of the terms “a” and “an” and “the” and similar terms in the context of describing embodiments of invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. In addition to the order detailed herein, the methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate embodiments of invention and does not necessarily impose a limitation on the scope of the invention unless otherwise specifically recited in the claims. No language in the specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

## Claims

1. An array that comprises a first plurality of dimeric probes that hybridize to a first target nucleic acid sequence, wherein each of the dimeric probes comprise a first hybridizing nucleic acid sequence and a second hybridizing nucleic acid sequence linked together, wherein the first and second hybridizing nucleic acid sequences are the same and hybridize to the first target nucleic acid sequence, and wherein the first plurality of dimeric probes are selected from SEQ ID NO:4 to SEQ ID NO:9, SEQ ID NO:19 to SEQ ID NO:24, SEQ ID NO:28 to SEQ ID NO:33, SEQ ID NO:42 to SEQ ID NO:45, SEQ ID NO:48 to SEQ ID NO:51, SEQ ID NO:57, SEQ ID NO:58 and SEQ ID NO:73 to SEQ ID NO:301.

2. The array of claim 1, wherein the first and second hybridizing nucleic acid sequences are linked directly together.

3. The array of claim 1, wherein the first and second hybridizing nucleic acid sequences are linked together via a nucleic acid linker sequence.

4. The array of claim 3, wherein the nucleic acid linker sequence is a poly-adenine linker.

5. The array of claim 1, wherein the dimeric probes are about 40-60 nucleotides in length.

6. The array of claim 5, wherein the dimeric probes are about 40-48 nucleotides in length.

7. The array of claim 5, wherein the dimeric probes are about 50-58 nucleotides in length.

8. The array of claim 1, wherein the array further comprises a second plurality of dimeric probes that hybridize to a second target nucleic acid sequence, wherein the second target nucleic acid sequence is from a fungal, viral, or bacterial pathogen.

9. The array of claim 8, wherein the first and second pluralities of dimeric probes hybridize to target nucleic acid sequences in the same pathogen.

10. The array of claim 8, wherein the first and second pluralities of dimeric probes hybridize to target nucleic acid sequences in different pathogens.

11. The array of claim 8, wherein the array further comprises more than two pluralities of dimeric probes that hybridize to different target nucleic acid sequences.

12. The array of claim 11, wherein the more than two pluralities of dimeric probes hybridize to target nucleic acid sequences in the same pathogen.

13. The array of claim 11, wherein the more than two pluralities of dimeric probes hybridize to target nucleic acid sequences in different pathogens.

14. The array of claim 1, wherein the array further comprises pluralities of dimeric probes that specifically hybridize to target nucleic acid sequences in about 50 different pathogens.

15. The array of claim 1, wherein the array further comprises pluralities of dimeric probes that specifically hybridize to target Ob4Anucleic acid sequences in about 100 different pathogens.

16. The array of claim 1, wherein the first target nucleic acid sequence is from Rhizoctonia solani, Pythium aphanidermatum, Fusarium solani or F. oxysporum.