Patent Application
20140271560
The instant application contains a Sequence Listing which has been submitted via EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Mar. 12, 2014, is named 1-55890_SL.txt, and is 53,393 bytes in size.
Head scab, also known as Fusarium head blight (FHB), is a devastating disease of wheat and barley that is primarily caused by the fungus Gibberella zeae (anamorph=Fusarium graminearum). This disease can reach epidemic levels and causes extensive damage to wheat and barley in humid and semi-humid wheat growing areas of the world. The disease has caused large scale devastation in the United States, Canada and China. Other countries of the world that produce large amounts of wheat in humid and semi-humid regions and would be susceptible to major outbreaks of FHB include India, Russia, France, Germany and the United Kingdom. FHB was responsible for almost 500 million bushels of wheat lost in the United States from 1991 until 2013. Economic loss has been estimated at 1.3 to 2.6 billion during this time period. The importance of FHB was recognized by the 105th U.S. Congress when it adopted the “Wheat and Barley Protection Act” that authorized expenditure of 26 million dollars for the study of FHB.
A number of different strategies have been employed to manage and control plant pathogens such as FHB. Chemical pesticides are often relied on to help control these pathogens. However, the widespread use of chemicals in agriculture has been a subject of growing public concern and scrutiny due to the potentially harmful effects on the environment and human health. Other problems linked to pesticide use, including the emergence of pesticide-resistant pathogens, have led to a gradual elimination of some available pesticides. In addition, there presently exist strict regulations on chemical pesticide use, and political pressure aiming to remove the most hazardous chemicals from the market. As a result, some chemical companies have become increasingly reluctant to develop and test new chemicals due to the concerns relating to registration process and cost. Alternatively, the practice of conventional tillage has been used against FHB. None of these approaches have led to substantial and consistent control of FHB. The residues and high cost of chemical fungicides and the desires for reduced or conservation tillage to prevent soil erosion and runoff discourage the use of these strategies.
There is a clear need for the development of non-chemical alternative methods to control plant pathogenic diseases, such as FHB. Biological control of plant pathogens has been increasingly considered a viable alternative to manage plant diseases. Particularly, the use of biologically active agents in the control of plant pests and pathogenic diseases has become especially important for certified organic growers who do not use synthetic chemicals for pest management.
Accordingly, disclosed herein are methods of using Cryptococcus flavescens strains to suppress FHB on cereal plants.
Disclosed are methods of identifying subspecies of Cryptococcus flavescens and methods of treating or suppressing Fusarium head blight with the different Cryptococcus flavescens strains. In particular, two genotypes, Genotypes A and B, were identified using the disclosed real time PCR technique. The following Cryptococcus flavescens strains were identified as being either Genotype A or B and as being able to suppress Fusarium head blight: Y-7373, YB-601, YB-602, Y-7377, Y-7372, Y-7375, Y-7374, Y-7376, YB-328, Y-7379, and YB-744.
In a particular embodiment described herein is a method for suppressing Fusarium head blight in a cereal plant comprising: a) identifying a genotype for a Cryptococcus flavescens strain by qPCR and/or by sequence identity; and b) applying to a seed head of said plant an effective amount of one or more microbial antagonists, wherein the one or more microbial antagonist is a Genotype A Cryptococcus flavescens strain or Genotype B Cryptococcus flavescens strain, as identified in step a), wherein the one or more microbial antagonists is not Cryptococcus flavescens 3C, which has been deposited under NRRL accession no. Y-50378, or Cryptococcus flavescens 4C, which has been deposited under NRRL accession no. Y-50379.
In another embodiment described herein, the genotype for a Cryptococcus flavescens strain is determined by quantitative PCR (qPCR), comprising the steps: a) performing qPCR on extracted genomic DNA utilizing one or more primer pairs sharing at least 90% sequence identity with the primer pairs of Table 1 (SEQ ID NOs: 45-72), wherein sequence identity is determined for each individual forward or reverse primer; and b) identifying a Cryptococcus flavescens strain as Genotype A or Genotype B, wherein: (1) the Cryptococcus flavescens strain is identified as Genotype A when qPCR results in: a threshold cycle value of 17-21 when using the btub.1, btub.2, or EF1.2 primer sets; a threshold cycle value of 18-20 when using the h22.1, h30.2, or h31.1 primer sets; a threshold cycle value of 17-20 when using the h31.2 primer set; or a threshold cycle value of 12-15 when using the 12.1 primer set; and (2) the Cryptococcus flavescens strain is identified as Genotype B when qPCR results in a threshold cycle value of 31-33 when using the btub.1 primer set; a threshold cycle value of 32-35 when using the btub.2 primer set; a threshold cycle value of 24-26 when using the EF1.2 primer set; a threshold cycle value of 22-24 when using the h22.1 or h31.1 primer sets; a threshold cycle value of 22-23 when using the h30.2 primer set; a threshold cycle value of 34-35 when using the h31.2 primer set; or a threshold cycle value of 15-18 when using the I2.1 primer set.
In another embodiment described herein, the genotype for a Cryptococcus flavescens strain is determined by sequence identity, comprising the steps: a) sequencing extracted genomic DNA of a target Cryptococcus flavescens; b) comparing the sequence determined in step a) to known homologous sequences of other Cryptococcus flavescens strains; and c) identifying a Cryptococcus flavescens strain as Genotype A or Genotype B, wherein: (1) the Cryptococcus flavescens strain is identified as Genotype A when the sequence of the extracted genomic DNA of the target Cryptococcus flavescens shares: 100% sequence identity at the β-tubulin gene to SEQ ID NO:1; between 95% and 100% sequence identity at the chitin synthase 1 gene to SEQ ID NOs: 2, 3, 4, 5, or 6; between 99% and 100% sequence identity at the EF1 gene to SEQ ID NOs: 7, 8, 9, 10, or 11; between 99% and 100% sequence identity at the hsp70 gene to SEQ ID NOs: 12, 13, 14, 15, or 16; or between 99% and 100% sequence identity at the cs22 target locus to SEQ ID. NOs: 85, 86, 87, 88, or 89; and (2) the Cryptococcus flavescens strain is identified as Genotype B when the sequence of the extracted genomic DNA of the target Cryptococcus flavescens shares: between 99% and 100% sequence identity at the β-tubulin gene to SEQ ID NOs: 17, 18, 19, 20, 21, 22 or 23; between 97% and 100% sequence identity at the chitin synthase 1 gene to SEQ ID NOs: 24, 25, 26, 27, 28, 29 or 30; between 99% and 100% sequence identity at the EF1 gene to SEQ ID NOs: 31, 32, 33, 34, 35, 36, or 37; between 99% and 100% sequence identity at the hsp70 gene to SEQ ID NOs: 38, 39, 40, 41, 42, 43 or 44; or 100% sequence identity at the cs22 target locus to SEQ ID NO: 90.
In another embodiment described herein, the extracted genomic DNA is collected from one or more cereal grass plants prior to application of one or more microbial antagonists to the one or more cereal grass plants.
In another embodiment described herein, the extracted genomic DNA is collected from one or more cereal grass plants following application of one or more microbial antagonists to the one or more cereal grass plants.
In another embodiment described herein, the extracted genomic DNA is collected from an in vitro Cryptococcus flavescens culture.
In another embodiment described herein, at least one of the one or more microbial antagonists is a Genotype A Cryptococcus flavescens strain.
In another embodiment described herein, at least one of the one or more microbial antagonists is a Genotype B Cryptococcus flavescens strain.
In another embodiment described herein, at least one of the one or more microbial antagonists is selected from the group of C. flavescens strains consisting of: Y-7373; YB-601; YB-602; Y-7377; Y-7372; Y-7375; Y-7374; Y-7376; YB-328; Y-7379; and YB-744.
In another embodiment described herein, at least one of the one or more microbial antagonists is tolerant to prothioconazole.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7373.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens YB-601.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens YB-602.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7377.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7372.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7375.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7374.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7376.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens YB-328.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens Y-7379.
In another embodiment described herein, at least one of the one or more microbial antagonist is C. flavescens YB-744.
In another embodiment described herein, in addition to the one or more microbial antagonists being applied to a seed head of a cereal plant, Cryptococcus flavescens 3C, which has been deposited under NRRL accession no. Y-50378, and/or Cryptococcus flavescens 4C, which has been deposited under NRRL accession no. Y-50379, is applied to the cereal plant.
In another embodiment described herein, the one or more microbial antagonists are applied to the seed head prior to a hard dough stage of development.
In another embodiment described herein, the one or more microbial antagonists are applied to the seed head during flowering.
In another embodiment described herein, the one or more microbial antagonists are applied to the seed head prior to flowering.
In another embodiment described herein, the cereal plant is wheat or barley.
In another embodiment described herein, the cereal plant is wheat.
In another embodiment described herein, the effective amount of at least one microbial antagonist is an amount sufficient to reduce the level of Fusarium head blight relative to that in a corresponding untreated control.
In another embodiment described herein, application of an effective amount of one or more microbial antagonists to the seed head of a cereal plant comprises spraying the one or more microbial antagonists onto the cereal plant.
In another embodiment described herein, the method of spraying is selected from the group consisting of: spraying through a sprinkler irrigation system; aerial spray application; ground-based spray application.
In another embodiment described herein, the effective amount of the one or more microbial antagonists is between about 104-109 CFU/ml applied at a rate of about 105-106 CFU/cm2.
In another embodiment described herein, the effective amount of the one or more microbial antagonists is about 1.5×109 CFU/ml applied at a rate of about 2×106 to 6×106 CFU/cm2.
In another embodiment described herein, the effective amount of the one or more microbial antagonists is about 2.3×108 CFU/ml applied at a rate of about 2×105 CFU/cm2.
In another embodiment described herein, the effective amount of the one or more microbial antagonists is about 3×108 CFU/ml at a rate of about 106 CFU/cm2.
In another embodiment described herein, application of an effective amount of the one or more microbial antagonists to the seed head of a cereal plant occurs at temperatures between about 5 to 35° C.
In another embodiment described herein, application of an effective amount of the one or more microbial antagonists to the seed head of a cereal plant occurs at temperatures between about 15 to 30° C.
In another embodiment described herein, the one or more microbial antagonists are substantially biologically pure.
In another embodiment described herein, two or more microbial antagonists are applied to the seed head of the cereal plant.
In another embodiment described herein, the two or more microbial antagonists are applied simultaneously.
In another embodiment described herein, the two or more microbial antagonists are applied separately.
In another embodiment described herein, at least two microbial antagonists are Genotype A Cryptococcus flavescens strains.
In another embodiment described herein, at least two microbial antagonists are Genotype B Cryptococcus flavescens strains.
In another embodiment described herein, a first microbial antagonist is a Genotype A Cryptococcus flavescens strain and a second microbial antagonist is a Genotype B Cryptococcus flavescens strain.
In another embodiment described herein, at least one microbial antagonist is selected from a first group consisting of: Genotype A Cryptococcus flavescens strains; and Genotype B Cryptococcus flavescens strains, and at least one microbial antagonist is selected from a second group consisting of: Cryptococcus flavescens 3C, which has been deposited under NRRL accession no. Y-50378; and Cryptococcus flavescens 4C, which has been deposited under NRRL accession no. Y-50379.
In another embodiment described herein, the method for suppressing Fusarium head blight in a cereal plant further comprises applying one or more fungicides to the cereal plant.
In another embodiment described herein, the fungicide is prothioconazole.
In another embodiment described herein, the one or more fungicides are applied to the cereal plant at a time selected from the group consisting of: prior to application of the one or more microbial antagonists; simultaneously with the application of the one or more microbial antagonists; and subsequent to the application of the one or more microbial antagonists.
In a particular embodiment described herein is a kit comprising one or more microbial antagonists disclosed herein.
In a particular embodiment described herein is a kit comprising one or more primer pairs sharing at least 90% sequence identity with the primer pairs of Table 1 (SEQ ID NOs: 45-72), wherein sequence identity is determined for each individual forward or reverse primer.
In a particular embodiment described herein is a method for identifying the genotype of a Cryptococcus flavescens strain comprising: a) performing qPCR on extracted genomic DNA utilizing one or more primer pairs sharing at least 90% sequence identity with the primer pairs of Table 1 (SEQ ID NOs: 45-72), wherein sequence identity is determined for each individual forward or reverse primer; and b) identifying a Cryptococcus flavescens strain as Genotype A or Genotype B, wherein: (1) the Cryptococcus flavescens strain is identified as Genotype A when qPCR results in: a threshold cycle value of 17-21 when using the btub.1, btub.2, or EF1.2 primer sets; a threshold cycle value of 18-20 when using the h22.1, h30.2, or h31.1 primer sets; a threshold cycle value of 17-20 when using the h31.2 primer set; or a threshold cycle value of 12-15 when using the I2.1 primer set; and (2) the Cryptococcus flavescens strain is identified as Genotype B when qPCR results in a threshold cycle value of 31-33 when using the btub.1 primer set; a threshold cycle value of 32-35 when using the btub.2 primer set; a threshold cycle value of 24-26 when using the EF1.2 primer set; a threshold cycle value of 22-24 when using the h22.1 or h31.1 primer sets; a threshold cycle value of 22-23 when using the h30.2 primer set; a threshold cycle value of 34-35 when using the h31.2 primer set; or a threshold cycle value of 15-18 when using the I2.1 primer set.
In a particular embodiment described herein is a method for identifying the genotype of a Cryptococcus flavescens strain comprising: a) sequencing extracted genomic DNA of a target Cryptococcus flavescens; b) comparing the sequence determined in step a) to known homologous sequences of other Cryptococcus flavescens strains; and c) identifying a Cryptococcus flavescens strain as Genotype A or Genotype B, wherein: (1) the Cryptococcus flavescens strain is identified as Genotype A when the sequence of the extracted genomic DNA of the target Cryptococcus flavescens shares: 100% sequence identity at the β-tubulin gene to SEQ ID NO:1; between 95% and 100% sequence identity at the chitin synthase 1 gene to SEQ ID NOs: 2, 3, 4, 5, or 6; between 99% and 100% sequence identity at the EF1 gene to SEQ ID NOs: 7, 8, 9, 10, or 11; between 99% and 100% sequence identity at the hsp70 gene to SEQ ID NOs: 12, 13, 14, 15, or 16; or between 99% and 100% sequence identity at the cs22 target locus to SEQ ID. NOs: 85, 86, 87, 88, or 89; and (2) the Cryptococcus flavescens strain is identified as Genotype B when the sequence of the extracted genomic DNA of the target Cryptococcus flavescens shares: between 99% and 100% sequence identity at the β-tubulin gene to SEQ ID NOs: 17, 18, 19, 20, 21, 22 or 23; between 97% and 100% sequence identity at the chitin synthase 1 gene to SEQ ID NOs: 24, 25, 26, 27, 28, 29 or 30; between 99% and 100% sequence identity at the EF1 gene to SEQ ID NOs: 31, 32, 33, 34, 35, 36, or 37; between 99% and 100% sequence identity at the hsp70 gene to SEQ ID NOs: 38, 39, 40, 41, 42, 43 or 44; or 100% sequence identity at the cs22 target locus to SEQ ID NO: 90.
Throughout this disclosure, various publications, patents and published patent specifications are referenced. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this invention pertains.
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided herein can be different from the actual publication dates, which can require independent confirmation.
It is to be understood that the present compositions and methods are not limited to specific synthetic methods unless otherwise specified, or to particular reagents unless otherwise specified, as these methods and reagents may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting. Moreover, it is to be understood that unless otherwise expressly stated, it is in no way intended that any method set forth herein be construed as requiring that its steps be performed in a specific order. Accordingly, where a method claim does not actually recite an order to be followed by its steps or it is not otherwise specifically stated in the claims or descriptions that the steps are to be limited to a specific order, it is in no way intended that an order be inferred, in any respect. This holds for any possible non-express basis for interpretation, including matters of logic with respect to arrangement of steps or operational flow, plain meaning derived from grammatical organization or punctuation, and the number or type of aspects described in the specification.
As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a C. flavescens strain,” “an amount,” or “the plant” includes mixtures of two or more such C. flavescens strains, amounts, or plants, and the like.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint and independently of the other endpoint. It is also understood that there are a number of values disclosed herein and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. It is also understood that each unit between two particular units is also disclosed. For example, if a range of 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.
The term “cereal” as used herein is intended to refer to any cereal plant species that is susceptible to FHB. Cereals reported to be susceptible to FHB include, but are not limited to, wheat, barley, rice, oats, spelt, and triticale, though wheat and barley are the two crops in which this disease presents a significant economic problem. Any of these cereals can be treated for FHB using the methods described herein.
The terms “microbial antagonist” refers to microorganisms that work to prevent, suppress, treat, or control the development of a pathogen or a pathogenic disease in a plant, such as a cereal plant. A microbial antagonist can work to prevent, suppress, treat, or control a pre-harvest or post-harvest disease in plants, including their fruits and other harvestable parts. For example, a microbial antagonist can be used to treat FHB. A microbial antagonist activity can be achieved by a variety of mechanisms. In some instances, the microbial antagonist can be antagonistic to, for example, a plant pathogen, such as FHB. An antagonist can itself be a bacterium, a fungus, or other type of microorganism. For example a microbial antagonist exhibits a degree of inhibition of FHB exceeding, at a statistically significant level, that of an untreated control.
The term “suppress” or “suppressing” refers to a reduction or inhibition. For example, “suppressing Fusarium head blight” refers to reducing the level of disease caused by Fusarium head blight. “Suppressing” Fusarium head blight is interchangeable with “treating” Fusarium head blight. To treat refers to the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. Thus, the term covers any treatment including preventing, inhibiting, suppressing or relieving the disease.
As used herein, the terms “effective amount” and “amount effective” refer to an amount of a composition that is sufficient to achieve a desired result or to have an effect on an undesired condition. For example, the effective amount can reduce the level of Fusarium head blight relative to that in the corresponding untreated control. An effective amount is a sufficient amount to achieve a desired result without resulting in undesired effects. In one aspect of the disclosed methods, the effective amount can vary depending on factors such as, but not limited to, the type of cereal plant being treated, the quantity, the route of administration, the duration of treatment, and any compositions or microbial antagonists used in combination.
As used herein, the term “percent identity” as applied to polynucleotide sequences refers to the percentage of residue matches between at least two sequences aligned using a standardized algorithm such as the ClustalW and Muscle algorithms employed by the Molecular Evolutionary Genetics Analysis (MEGA) software (Center for Evolutionary Medicine and Informatics, Tempe, Ariz.). Other alignment tools may be used to determine the percent identity, such as the BLAST suite of programs (National Center for Biotechnology Information) (e.g., blast, blastp, blastx, nucleotide blast and protein blast) using, for example, default parameters.
Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides. Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
As used herein, the terms “homologue” and “homologous” when used to describe a sequence refers to a sequence that is a variant of another and wherein the two sequences are evolutionarily related. In the present disclosure, when a particular gene is referred to, the term is meant to encompass homologues and orthologues, variants, derivatives, and mutants of such a gene or protein. The present invention is not limited to embodiments employing the exact sequence of any of the disclosed polynucleotides, but encompasses any variant that is related by structure, sequence, function or is derived in any way from the named polynucleotide. For example, the present invention encompasses polynucleotides having, for example, at least 85% primary nucleic acid sequence identity among all strains of C. flavescens for genes β-tublulin, chitin synthase 1, elongation factor 1, heat shock protein 70 kDa, and cs22 (chitin synthase 5 plus downstream anonymous region).
“Variant” refers to a polynucleotide that differs from a reference polynucleotide, but retains essential properties. A typical variant of a polynucleotide differs in nucleic acid sequence from another, reference polynucleotide. Generally, differences are limited so that the sequences of the reference polynucleotide and the variant are closely similar overall and, in many regions, identical. A variant and reference polynucleotide may differ in nucleic acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A variant may be naturally occurring, or it may be a variant that is not known to occur naturally. A polynucleotide variant may have, for example, at least 99%, 95%, 90%, or at least 85% percent identity over the entire length of the original polynucleotide. Variants may be derivatives of the polynucleotide of which they are a variant, they may be chemically or biochemically modified and have one or more amino nucleotide substitutions, additions, and/or deletions. Variants may share certain functionally significant motifs with the polynucleotide of which they are a variant.
As used herein, the term “threshold cycle (Ct)” refers to the intersection between a qPCR amplification curve and a threshold line. The threshold line is commonly ten times the standard deviation of the background fluorescence. The first cycle which is above the threshold line is defined as the Ct. Within a certain range of template concentrations, the Ct value is proportional to the template copy number present at the beginning of the reaction and reflects the first opportunity for quantification of the template. Mismatches in the primer-probe sequences disclosed herein result in altered efficiency of DNA amplification. Generally, an increased number of mismatches leads to lowered qPCR efficiency. It is these altered qPCR efficiencies (and the underlying primer-probe mismatches) that allow for discrimination between Genotype A and Genotype B C. flavescens, as disclosed herein.
Methods of identifying specific genotypes of C. flavescens are provided. For example, Genotype A and Genotype B C. flavescens strains can be identified using the disclosed quantitative PCR or sequence identity methods. Cryptococcus is a genus of fungus that grows in culture as yeasts. Cryptococcus flavescens, a species of Cryptococcus, is an encapsulated yeast. C. flavescens is a biological control agent of FHB and has been shown to have improved desiccation tolerance. Different genotypes of C. flavescens can treat diseases, such as FHB, with different efficiencies. Therefore, it can be advantageous to develop methods of distinguishing between the C. flavescens genotypes.
Real time PCR, also referred to as quantitative PCR (qPCR), can be used to identify different species or genotypes of C. flavescens. The qPCR assay is faster, cheaper, and more reliable than the previous detection methods based solely on culturing and antibiotic resistance of the target strain. The primers and assay conditions can be readily adapted to isolate environmental strains of C. flavescens that are genetically similar to 3C and express similar biocontrol phenotypes. Furthermore, additional primer sets can be defined using the genome sequence provided by GenBank accession numbers CAUG01000001-CAUG01000712. For example, a C. flavescens Genotype A or Genotype B strain can be identified by performing qPCR on DNA obtained from a microorganism. The qPCR results in a threshold cycle value for each strain. Depending on the primer set used during qPCR, the threshold cycle value can be indicative of the specific C. flavescens genotype. Described below are examples of qPCR primers and threshold cycle values that are indicative of Genotype A and Genotype B C. flavescens strains.
Another method of identifying C. flavescens genotypes includes determining the sequence identity of specific genes. For example, determining the sequence identity of one or more of the (3-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70 kDa, and cs22 (chitin synthase 5 plus downstream anonymous region) gene sequences and comparing the determined sequence with the homologous gene sequences in known Genotype A and Genotype B C. flavescens strains can be used to identify the genotype of the strain being tested. A multilocus sequence analysis of β-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70, and cs22 (chitin synthase 5 plus downstream anonymous region) can also be used to classify C. flavescens strains into two distinct groups, or genotypes, as well. Some regions of the C. flavescens DNA, such as the Internal Transcribed Spacer (ITS) region, cannot be used to differentiate the C. flavescens subspecies because all C. flavescens strains have the same ITS sequence. Examples of sequence identity being used to determine Genotype A and Genotype B. C. flavescens strains are described below.
As described herein, threshold cycle values determined during qPCR or sequence identity can be used to differentiate the genotypes of C. flavescens. These strains can be used in the disclosed methods. The methods of identifying can include the initial step of collecting the DNA to be tested. For example, genomic DNA can be extracted from a microorganism, or cereal plant seed heads can be collected and the DNA isolated from these heads. Once the DNA has been obtained, qPCR can be performed using the primers and probes identified herein or the DNA can be sequenced, using known sequencing techniques, in order to determine the sequence identity when compared to known C. flavescens strains.
In some aspects, the methods of identifying genotypes of C. flavescens include performing qPCR with more than one primer set. The methods can also include comparing the sequence identity at more than one gene. In some instances, the methods of identifying can incorporate both the qPCR technique and the sequence identity technique for identifying a Genotype A or Genotype B C. flavescens strain. For example, qPCR using a particular primer set can be performed on DNA isolated from a microorganism and comparing the sequence identity of a particular gene of the isolated DNA to known Genotype A and Genotype B strains can also be performed. Therefore, the combination of the qPCR and the sequence identity results can provide the identity of the C. flavescens genotype.
Strains of C. flavescens in addition to those identified herein can be identified and recovered by those of skill in the art using the primers and the sequence identity described herein.
The disclosed methods can include the use of the C. flavescens strains described herein as well as compositions containing the C. flavescens strains described herein.
i. Genotype A
C. flavescens Genotype A strains can be used for the disclosed methods. Examples of Genotype A strains include OH 182.9 (3C), Y-7373, Y7377, YB601 and YB-602.
Genotype A strains can be detected by qPCR. In one aspect, amplification is performed with one or more of the primer sets selected from btub.1, btub.2, h31.2, EF1.2,h22.1, h30.2,h31.1 and 12.1 (see Table 1 and
The Genotype A strains to be used in the methods described herein can also be identified by comparing a determined polynucleotide sequence to the known homologous sequences of other Genotype A strains. For example, the percent identity of one or more of the gene sequences of (3-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70, and cs22 (chitin synthase 5 plus downstream anonymous region) compared to the disclosed sequences for these genes can be used to identify a Genotype A strain. Those strains that share the sequence identities disclosed below are considered Genotype A strains.
a. β-Tubulin Gene Sequence.
The Genotype A strains share 100% sequence identity among the strains at the β-tubulin gene. Therefore, those C. flavescens strains that have 100% identity to SEQ ID NO: 1 can be classified as a Genotype A strain.
b. Chitin Synthase 1 Gene Sequence.
The Genotype A strains can share between 95% and 100% sequence identity at the chitin synthase 1 gene. In one aspect, Genotype A strains share at least 95.2% sequence identity at the chitin synthase 1 gene. Therefore, C. flavescens strains that have between 95% to 100% sequence identity to SEQ ID NOs: 2, 3, 4, 5, or 6 can be classified as a Genotype A strain.
c. Elongation Factor 1 (EF1) Gene Sequence.
The Genotype A strains can share between 99% and 100% sequence identity at the EF1 gene. In one aspect, Genotype A strains share at least 99.7% sequence identity at the EF1 gene. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 7, 8, 9, 10, or 11 can be classified as a Genotype A strain.
d. Heat Shock Protein 70 kDa (Hsp70) Gene Sequence
The Genotype A strains can share between 99% and 100% sequence identity at the hsp70 gene. In one aspect, Genotype A strains share at least 99% identity at the hsp70 gene. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 12, 13, 14, 15, or 16 can be classified as a Genotype A strain.
e. Cs22 (Chitin Synthase 5 Plus Downstream Anonymous Region) Target Locus Sequence.
The Genotype A strains can share between 99% and 100% sequence identity at the cs22 target locus. In one aspect, Genotype A strains share at least 99% identity at the cs22 target locus. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 85, 86, 87, 88, or 89 can be classified as a Genotype A strain.
ii. Genotype B
C. flavescens Genotype B, or non-3C, strains can be used for the disclosed methods. Examples of Genotype B strains include Y-7372, Y-7374, Y-7375, Y-7376, Y-7379, YB-328, and YB-744.
Genotype B strains can be detected by qPCR. In one aspect, amplification is performed with one or more of the primer sets selected from btub.1, btub.2, h31.2, EF1.2, h22.1, h30.2, h31.1 and 12.1 (see Table 1 and
The Genotype B strains to be used in the methods described herein can also be identified by comparing a determined polynucleotide sequence to the known homologous sequences of other Genotype B strains. For example, the percent identity of one or more of the gene sequences of (3-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70, and cs22 (chitin synthase 5 plus downstream anonymous region) compared to the disclosed sequences for these genes can be used to identify a Genotype B strain. Those strains that share the sequence identities disclosed below are considered Genotype B strains
a. β-Tubulin Gene Sequence
The Genotype B strains can share between 99% and 100% sequence identity at the (3-tubulin gene. In one aspect, Genotype B strains share at least 99.6% sequence identity at the (3-tubulin gene. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 17, 18, 19, 20, 21, 22 or 23 can be classified as a Genotype B strain.
b. Chitin Synthase 1 Gene Sequence
The Genotype B strains can share between 98% and 100% sequence identity at the chitin synthase 1 gene. In one aspect, Genotype B strains share at least 98.8% sequence identity at the chitin synthase 1 gene. C. flavescens strains that have between 97% and 100% sequence identity to SEQ ID NOs: 24, 25, 26, 27, 28, 29 or 30 can be classified as a Genotype B strain.
c. Elongation Factor 1 Gene Sequence
The Genotype B strains can share between 99% and 100% sequence identity at the EF1 gene. In one aspect, Genotype B strains share 99.7% sequence identity at the EF1 gene. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 31, 32, 33, 34, 35, 36, or 37 can be classified as a Genotype B strain.
d. Heat Shock Protein 70 kDa Gene Sequence
The Genotype B strains can share between 99% and 100% sequence identity at the hsp70 gene. In one aspect, Genotype B strains share 99.1% sequence identity at the hsp70 gene. C. flavescens strains that have between 99% and 100% sequence identity to SEQ ID NOs: 38, 39, 40, 41, 42, 43 or 44 can be classified as a Genotype B strain.
e. Cs22 (Chitin Synthase 5 Plus Downstream Anonymous Region) Gene Sequence.
The Genotype B strains share 100% sequence identity among the strains at the cs22 target locus. Therefore, those C. flavescens strains that have 100% identity to SEQ ID NO: 90 can be classified as a Genotype B strain.
iii. Identifying a Fungus as C. flavescens
Fungi may be identified as a strain of C. flavescens by comparing a determined polynucleotide sequence to the known homologous sequences of other C. flavescens strains. For example, the percent identity of one or more of the gene sequences of β-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70, and cs22 (chitin synthase 5 plus downstream anonymous region) compared to the disclosed sequences for these genes can be used to identify a fungus as a species of C. flavescens.
a. β-Tubulin Gene Sequence
The C. flavescens strains can share between 95% and 100% sequence identity at the β-tubulin gene. In one aspect, C. flavescens strains share at least 95.1% sequence identity at the β-tubulin gene.
b. Chitin Synthase 1 Gene Sequence
The C. flavescens strains can share between 89% and 100% sequence identity at the chitin synthase gene. In one aspect, C. flavescens strains share at least 95.5% sequence identity at the chitin synthase 1 gene.
c. Elongation Factor 1 Gene Sequence
The C. flavescens strains can share between 94% and 100% sequence identity at the elongation factor 1 gene. In one aspect, C. flavescens strains share at least 94.1% sequence identity at the elongation factor 1 gene.
d. Heat Shock Protein 70 kDa Gene Sequence
The C. flavescens strains can share between 93% and 100% sequence identity at the heat shock protein 70 gene. In one aspect, C. flavescens strains share at least 93.7% sequence identity at the heat shock protein 70 gene.
e. cs22 (Chitin Synthase 5 Plus Downstream Anonymous Region) Gene Sequence.
The C. flavescens strains can share between 79% and 100% sequence identity at the cs22 target locus. In one aspect, C. flavescens strains share at least 79.4% sequence identity at the cs22 target locus.
f. Identification of a Fungus as a Strain of C. flavescens
Across a concatenation of the genes β-tubulin, chitin synthase 1, elongation factor 1, heat shock protein 70, and cs22, C. flavescens strains share at least 89.8% sequence identity. Therefore, a fungus sharing at least 89.8% identity among these five genes may be identified as a species of C. flavescens.
Methods for suppressing FHB in a cereal plant by applying to a seed head of said plant an effective amount of at least one microbial antagonist are provided. The microbial antagonist can be a Genotype A C. flavescens strain or Genotype B Cryptococcus flavescens strain, wherein the strain of C. flavescens is not C. flavescens 3C which has been deposited under NRRL accession no. Y-50378 or C. flavescens 4C which has been deposited under NRRL accession no. Y-50379.
In one aspect of the methods, a Genotype A C. flavescens strain can be used to suppress FHB in a cereal plant by applying an effective amount of at least one Genotype A C. flavescens strain to a seed head of the cereal plant. Any of the Genotype A C. flavescens strains described herein can be used. For example, Genotype A C. flavescens strains can include Y-7373, Y7377, YB601 and YB-602.
In one aspect of the methods, a Genotype B C. flavescens strain can be used to suppress FHB in a cereal plant by applying an effective amount of at least one Genotype B C. flavescens strain to a seed head of the cereal plant. Any of the Genotype B C. flavescens strains described herein can be used. For example, Genotype B C. flavescens strains can include Y-7372, Y-7374, Y-7375, Y-7376, Y-7379, YB-328, and YB-744.
The disclosed methods involve applying an effective amount of C. flavescens to a cereal plant. An effective amount of C. flavescens can be an amount that reduces the level of FHB relative to that in a corresponding untreated control. An effective amount of C. flavescens can be from 104 to 109 CFU per ml. For example, C. flavescens levels between 107-108 CFU/ml can be used. In some instances, 5×109 CFU/ml can be applied at 20 gal/acre. In one aspect of the disclosed methods, the level of FHB in untreated controls is previously determined.
A. Fusarium Head Blight (FHB)
Head scab, also known as FHB, is a devastating disease of cereal plants, such as, wheat and barley, that is primarily caused by the fungus Gibberellazeae (anamorph=Fusarium graminearum). Fusarium graminearum primarily infects the heads (flower heads, seed heads, or spikes) of cereal plants from the time of flowering through the soft dough stage of head development. Germinated conidia or ascospores of Fusarium graminearum penetrate through anthers and associated tissues to initiate infection of the host and the development of symptoms of FHB.
The infection of seed by Fusarium graminearum reduces seed germination, seedling vigor and plant emergence [Bechtel et al., (1985) Cereal Chem. 62:191-197]. Infection of wheat kernels by Fusarium graminearum reduces grain yield and affects grain quality [Clear et al., (1990) Can. J. Plant Sci. 70:1057-1069]. In addition to causing grain yield loss, Fusarium graminearum can produce mycotoxins such as the estrogenic toxin zearalenone (F-2) (Hesseltine et al., 1978, Fungi, especially Gibberellazeae, and zearalenone occurrence in wheat. Mycologia, 70, 14-18) and the trichothecenedeoxynivalenol (DON, vomitoxin) (Snijders, 1990, Fusarium head blight and mycotoxin contamination of wheat, a review. Netherlands Journal of Plant Pathology, 96, 187-198) that can have a deleterious effect on grain quality [Cardwell et al., 2001, Mycotoxins: the cost of achieving food security and food quality, APS Net: Feature story August, 2001] and animal health [Marasas, 1991, Toxigenic Fusaria, in: Mycotoxins and Animal Foods, J. E. Smith and R. S. Henderson, eds., CRC Press, Inc., Boca Raton, Fla.; Beardall and Miller, 1994, Diseases in humans with mycotoxins as possible causes, in Mycotoxins in Grain: Compounds Other than Aflatoxin (MILLER, J. D. & TRENHOLM, H. L., Eds.). Eagan Press, St. Paul, Minn., pp. 387-39]. Therefore, there is a strong need for treating for FHB.
A common treatment of FHB comprises the application of the fungicide prothioconazole. Prothioconazole allows for an increase in grain yields and a reduction of DON in wheat kernels. However, the use of fungicides, such as prothioconazole, is becoming more and more unacceptable due to environmental concerns and possible side effects in humans or animals that consume products treated with fungicides. Although new FHB treatments may eventually replace prothioconazole, the possibility also exists that new treatments may be combined with prothioconazole treatment. Therefore, the disclosed microbial antagonists for FHB can be prothioconazole-tolerant.
B. Microbial Antagonists
The disclosed methods include the use of microbial antagonists that are strains of C. flavescens. Compositions containing the disclosed microbial antagonists are also disclosed. Although C. flavescens OH 182.9 (aka 3C) can be used to reduce FHB in cereals, it is sensitive even to low concentrations of the fungicide prothioconazole. This sensitivity has limited the application of C. flavescens against FHB in fields which are or will also be treated with prothioconazole. Variants, mutants, or other C. flavescens strains, which exhibit significantly greater tolerance to prothioconazole than the parent 3C, can be used in the disclosed methods. The prothioconazole tolerant strains can exhibit greater efficacy against FHB than the parent C. flavescens 3C strain.
Microbial antagonists useful in the methods described herein can be, but are not limited to, C. flavescens (Y-7373), C. flavescens (YB-601), C. flavescens (YB-602), C. flavescens (Y-7377), C. flavescens (Y-7372), C. flavescens (Y-7375), C. flavescens (Y-7374), C. flavescens (Y-7376), C. flavescens (YB-328), C. flavescens (Y-7379), or C. flavescens (YB-744). Other examples of C. flavescens strains can be found in U.S. Pat. No. 8,241,889. The strains described in these patents can be used in the disclosed methods to treat FHB.
The C. flavescens strains used to suppress FHB in cereal plants can be Genotype A or Genotype B C. flavescens strains. Therefore the microbial antagonists used in the disclosed methods can include the C. flavescens strains identified using the qPCR and sequence identity methods of identifying described above.
In some aspects, the microbial antagonists can be a substantially pure microorganism which is a strain of C. flavescens selected from the group consisting of Genotype A C. flavescens strain or Genotype B C. flavescens strain, wherein the strain of C. flavescens is not C. flavescens 3C which has been deposited under NRRL accession no. Y-50378 or C. flavescens 4C which has been deposited under NRRL accession no. Y-50379.
C. Cereal Plants
Cereal plants include any seed or plant that produces an edible seed, fruit or grain, including, but not limited to, oat, rye, wheat, rice, maize, sorghum, and millet. Wheat, barley, oats, spelt and triticale are known to be susceptible to FHB and can therefore be treated in the disclosed methods. Because cereal plants are consumed every day by both humans and livestock, it is critical to keep these plants from being contaminated with harmful agents, such as FHB, to the extent they cannot be used. Methods that prevent, inhibit or suppress the growth or harmful effects of agents such as FHB are considered to be methods that promote the healthy growth or life cycle of cereal plants.
D. Administration of the Microbial Antagonist
The microbial antagonists can be administered depending on a variety of factors. Administration can vary depending on factors, such as, but not limited to, stage of development of the cereal plant, weather, amount of microbial antagonist already present on the cereal plant, and the method of administration.
i. Stage of Development of Cereal Plant
The application of the microbial antagonist to the seed head can occur at any time after the boot stage and before the hard dough stage of cereal development. The cereal head can be susceptible to infection by Fusarium graminearum from the onset of flowering (anthesis) through the soft dough stage of kernel development. Thus, one time to apply the biological control agents would be from the time immediately preceding flowering until as late as the soft dough stage of kernel development. Application of antagonists to heads before flowering would allow antagonists to have colonized wheat head parts prior to the wheat head becoming susceptible to infection. Additionally, antagonists would be well positioned to colonize and protect anthers as they emerge from florets. The antagonists would still be effective if applied after flowering has begun, but before the hard dough stage of development. However, long delays may decrease the effectiveness of the treatment depending on methods of cell formulation and application. Therefore, application of the microbial antagonist can occur prior to hard dough stage of development, during flowering, or prior to flowering.
ii. Weather
The temperature at which the C. flavescens can be applied can vary. The microbial antagonists can be effective at temperatures ranging from about 5° C. to about 35° C. In one aspect of the disclosed methods, the microbial antagonists are applied at temperatures ranging from 15-30° C.
The wind or rain can also play a role in the administration of the microbial antagonists. For example, heavy wind conditions or rain may result in less microbial antagonist remaining on the cereal plant. If the wind or rain prevent some, or all, of the microbial antagonist from remaining on the cereal plant, the therapeutic effects can be minimal or non-existent.
iii. Presence of Microbial Antagonist on the Cereal Plant
Administration of microbial antagonists can also depend on whether the microbial antagonist is already present on the plant and in what quantity. Therefore, in one aspect of the methods, it may be helpful to first test the cereal plant to determine if any microbial antagonist, such as Cryptococcus flavescens is present. If C. flavescens is present, determining the quantity can be beneficial. If C. flavescens is present, but not in an amount that is considered to be an effective amount, then applying more C. flavescens to the crop may be necessary. If there is an effective amount already present on the crop then treatment with more C. flavescens can be delayed.
iv. Methods of Administration
The application of C. flavescens strains to cereal plants can vary. In one aspect, the microbial antagonists can be sprayed onto the plants. The spraying can occur through a sprinkler irrigation system or an aerial or ground-based applicator. The administration of microbial antagonists can vary depending on factors, such as, but not limited to, the size of the area receiving the antagonists and the weather. Those of ordinary skill in the art would understand how to apply microbial antagonists to cereal plants.
E. Combination Treatment
FHB can be suppressed using a combination treatment. The disclosed methods can include applying a combination of C. flavescens strains. The combination can include two or more Genotype A strains, two or more Genotype B strains or a mixture of one or more Genotype A and Genotype B strains.
In some aspects of the disclosed methods, the microbial antagonist can include a Genotype A or Genotype B C. flavescens strain in combination with C. flavescens 3C which has been deposited under NRRL accession no. Y-50378 or C. flavescens 4C which has been deposited under NRRL accession no. Y-50379. Thus, in one aspect, at least one of the C. flavescens strains is C. flavescens 3C or C. flavescens 4C and at least a one strain is not C. flavescens 3C or C. flavescens 4C.
A combination treatment can also include treating with a fungicide, such as prothioconazole, as well as a C. flavescens strain. Any of the disclosed C. flavescens strains can be used in the combination treatments. The order of the treatment can vary. In one instance, cereal plants are first treated with prothioconazole and then treated with one or more C. flavescens strains. In one instance the prothioconazole and C. flavescens strains are applied simultaneously. In yet another instance, the prothioconazole is applied after treatment with C. flavescens.
In certain aspects, cereal plants are treated with a first C. flavescens strain and then treated with one or more additional C. flavescens strains. In one aspect, the first C. flavescens strain is applied simultaneously with the additional C. flavescens strains. In another aspect, the additional one or more C. flavescens strains are applied in one or more applications subsequent to the application of the first C. flavescens strain.
Methods of determining the presence of C. flavescens, particularly a Genotype A or Genotype B C. flavescens strain, on a cereal plant are provided. Any of the disclosed qPCR methods or primers can be used to identify the presence of Genotype A or Genotype B C. flavescens strains. For example, qPCR can be performed on a sample taken from a cereal plant in the field. Using the same threshold cycle value analysis developed for identifying specific C. flavescens genotypes, the presence of Genotype A or Genotype B strains can be determined.
In one aspect, at least one Genotype A strain and at least one Genotype B strain are each present. If only a Genotype B strain is determined to be present on the cereal plant, then a Genotype A strain can be administered to the cereal plant. Of course, the presence of a C. flavescens strain alone may not be the only factor involved in determining what, if any, treatments are needed on the cereal plant. The quantity of the C. flavescens can also play a role. Thus, if there is not an effective amount of the C. flavescens present, then administration of a Genotype A or Genotype B strain may be necessary.
The presence of a Genotype A or Genotype B C. flavescens strain can also be determined based on the methods described herein regarding using the sequence identity of one or more particular genes in the sample compared to the gene sequence of a known Genotype A or B C. flavescens strains.
The method of determining the presence of a Genotype A or Genotype B C. flavescens strain can be performed before or after administration of one of the disclosed microbial antagonists. In one aspect of the methods, the presence of the C. flavescens strains can be determined both prior to and after administration of a microbial antagonist.
Therefore, the methods can be used to verify the presence of a microbial antagonist on the cereal plant. In one aspect of the methods, after determining the presence of a Genotype A C. flavescens strain, either more Genotype A strain can be administered or a Genotype B strain can be administered. In some instances, a combination of C. flavescens genotypes can be administered.
The methods of determining or verifying the presence of C. flavescens can include collecting a cereal plant seed head sample. Collecting the sample can be performed by an individual or a machine or device used in the field. Once the sample has been collected, the DNA can be harvested from the cereal plant and qPCR or sequencing described herein can be performed so that the threshold cycle value or the sequence identity to known C. flavescens genotypes can be used to determine the presence of a Genotype A or Genotype B C. flavescens strain.
Because certain strains and genotypes of C. flavescens have more beneficial effects on treating FHB than other strains, there is a need for determining the presence of C. flavescens on cereal plants, especially determining the genotype of the C. flavescens.
In one aspect of the methods, the presence of a fungicide, such as prothioconazole, is determined. If prothioconazole is determined to be present then using a prothioconazole tolerant strain of C. flavescens to treat the cereal plant can be necessary.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the compounds, compositions, and methods claimed herein are used and evaluated and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. Unless indicated otherwise, parts are parts by weight, temperature is in ° C. or is at ambient temperature, and pressure is at or near atmospheric.
Materials and Methods
Quantitative PCR (qPCR). Each qPCR reaction had a total volume of 25 μl, consisting of 2.5 μl template DNA, 0.4 pmol/μl of each primer (forward primer h31.2_F: 5′-CGTCAGCGTGTTGCCACTTCGT-3′ (SEQ ID NO: 67); reverse primer h31.2R: 5′-GCTGCTGTCTTGCGGTCGCTTA-3′ (SEQ ID NO: 68)), 12.5 μl iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc., California, USA) and template-free PCR water as the rest of the volume. Template-free PCR water was also used as the template of negative controls. The thermal cycling program was run on the iCycler and the fluorescence data was collected by the iQ™5 detection system (Bio-Rad Laboratories, Inc.). Amplification was two-step with 3 min at 95° C. followed by 40 cycles of 15 s at 95° C. and 1 min at 72° C. Then the melting curve was run from 55° C. to 95° C. with an increment of 0.5° C. and a dwelling time of 10 s at each temperature. The raw data of qPCR was analyzed using the iQ™5 Optical System Software (Bio-Rad Laboratories, Inc.).
Results
Specific qPCR primers can be used to subtype C. flavescens strains into two genotypes, A and B. The primers designed to test the C. flavescens strains are shown in Table 1. Of the 12 sets tested against 6 different C. flavescens strains, EF1.1 and 5I could not distinguish any variation in vitro. However, primer sets btub.1, btub.2 and h31.2 did distinguish 2 different genotypes by differences of 11 to 14 in threshold cycle value. Primer sets EF1.2, h22.1, h30.2, h31.1, and 12.1 also distinguished 2 different genotypes by differences of 1 to 3 in threshold cycle value.
The amplification curves of qPCR using the primers from Table 1 are shown in Figures. 3A-3J. These curves show the differences in threshold cycle value displayed by different genotypes.
The primers and target loci used for the multilocus sequence typing analysis (MLST) are shown in tables 2 and 3, respectively. All C. flavescens isolates were found to have ITS sequences (covering the ITS1+2 regions of the rRNA gene) identical to strain 3C and distinct from C. neoformans (
aThe numbers were approximate since the minority of the strains have insertion or deletion (less than 1% of the total numbers).
bThe 5.8S and ITS regions were counted here as exon and introns, respectively.
Two distinct genotypes of Cryptococcus flavescens can be identified using the primer sets described herein. Genotype A is defined by the type strain OH 182.9 (3C), as well as isolates Y-7273, YB601 and YB-602, all of which are readily detected by the qPCR assay (
The sensitivity and specificity of the qPCR assay was determined. The primer pair h31.2 used in the qPCR assay were designed to amplify part of a putative Heat Shock Protein 70 kDa (Hsp70) gene identified from the genomic sequence of 3C identified by accession numbers CAUG01000001-CAUG01000712. The target region was 150 bp and includes an exon-intron conjunction. The products of selected qPCR reactions were run on an electrophoresis gel to show the banding patterns (
aThe percentage of columns in the alignment for which all the sequences were identical.
bThe strains classified as 3C type are 3C, Y-7373, Y-7377, YB-601 and YB-602; those classified as non-3C type are Y-7372, Y-7374, Y-7375, Y-7376, Y-7379, YB-328 and YB-744.
There was also a lighter band of slightly >200 bp than the expected target region of 150 bp (
To test the specificity of this assay within the Cryptococcus flavescens species, qPCR was also run on the 1 ng/μl genomic DNA templates of six other C. flavescens strains than 3C (
The ability of genotype A and genotype B of C. flavenscens to suppress FHB on wheat was assessed in five separate greenhouse bioassays. Reduction in disease severity caused by pre-inoculation with C. flavescens strains were observed in four of five independent trials. In two of those trials, the observed pattern was statistically significant (P<0.10 by the K-W test) after 16 days of incubation. In total, the Genotype A strains displayed greater biocontrol activity than the B genotype strains in three of five trials.
Significant reductions in disease severity due to inoculation by C. flavescens were observed at 10 and 16 days post-inoculation (P=0.11 and 0.03, respectively, by the K-W test;
Materials and Methods
Strains. Cryptococcus flavescens strain OH 182.9 (3C) was obtained from the Northern Regional Research Laboratory (NRRL) under accession number NRRL Y-30216. Six other C. flavescens strains were also obtained from NRRL under accession numbers Y-7372, Y-7373, YB-328, YB-601, YB-602 and YB-744, respectively. All the strains were maintained as glycerol stocks at −80° C. for long term storage.
Field establishment and 3C application. The experiments of this study were performed in two fields planted with Soft red winter wheat (Triticumaestivum L.) at the Snyder Farm at the northern end of Wooster, Ohio. The first field, subsequently noted as Field One, was planted with Cultivar Hopewell on Oct. 11, 2010. The layout of Field One is shown in
Log-phase cells of 3C produced in SDCL medium served as a 5% seed inoculum for the production in B Braun D-100 fermentor charged with 80 liters of SDCL medium. The fermentor was operated at 25° C., 20 liter/min aeration and 200 revolution/min with agitation provided by twin Rushton impellers. Eighteen hours after inoculation, fermentor temperature was reduced to 15° C. to cold shock the cells for 24 h prior to harvest. The cells were harvested through concentration into a paste using a Sharples 12-V tubular bowl centrifuge. The cell paste was then suspended in weak PO4 buffer at a concentration of approximately 2×109 CFU/ml and frozen at −20° C. as stock. The field inoculum was prepared right before application by mixing 1.5 liter of stock (pre-thawed at 10° C. overnight) with 1.5 liter sterile double distilled water and 10.8 ml 10% Tween 80. During anthesis of the wheat (Feekes Stage 10.5; on May 29, 2011), three areas of 6.1 m by 6.1 m (Plots i—1, i—2, and i—3 of FIG. S1) were sprayed with the 3C inoculum of approximately 1.5×109 cells/ml at the rate of 2×106 to 6×106 cells per square centimeter.
The method of producing 3C inoculum for Field Two was similar to that in Schisler et al. (Plant Disease, 2002). Briefly, 500-ml flasks containing 200 ml SDCL medium inoculated with 3C (starting OD of 0.10 at A620) were incubated at 25° C. at 250 revolution/min for 24 h. These cultures were then used to seed 3-L Fernbach flasks containing 1.25 L of SDCL medium to an OD of 0.10. Cultures were incubated for 48 h at 25° C. and 250 revolution/min. Colonized broth of 2.3×108 CFU/ml was then transferred into sterile containers, transported to the field on ice and applied at the rate of 4.4×105 CFU per square centimeter in the field within 24 h. The application was performed during anthesis of the wheat (Feekes Stage 10.5; on May 29, 2012). Sampling for this study was performed in 12 plots, 6 of which were sprayed with 3C only and another 6 of which were not sprayed with any microorganism. Some other plots in the field were also sprayed with 3C but they were all sprayed with other microorganism as well. The plot locations of different treatments were random designed across the field.
Sample collection. Wheat head samples were collected by clipping at the stalk 1 to 2 cm below the head and sealing each head in an individual sample bag. During sample collection, each head was only touched with the inside of the bag used to collect it and the clipping scissors were sterilized with 75% aqueous ethanol to prevent cross-contamination. Heads were collected from Field One immediately after the spray application of 3C (0 day post inoculation) as well as 24 h (1 day), 10, 26 and 44 days post inoculation in the summer of 2011 (see
Postharvest residue samples were collected from the three inoculated areas of Field One in late January and late March of 2012. Seven different locations were sampled within a circle of about 1-m radius around the center of each inoculated area. At each time point, two types of samples were collected from each location: a combination of soil, living weeds and brownish rotting plant materials was collected as one composite sample, and non-rotting wheat stalks were collected as the other sample. The grains were harvested from each plot as a whole for the 12 plots of Field Two on Jun. 26, 2012. The total grains of each plot were stored in one paper bag as a layer of thinner than 6 cm at dark at room temperature. One subsample of approximately 10 g was collected from the total grains of each plot immediately prior to grinding up for DNA extraction at 14, 64 and 155 days postharvest, respectively.
Sample handling, DNA extraction, and template preparation. Genomic DNA of 3C and the six other C. flavescens strains was extracted from 2-day old cultures grown on ⅕ Tryptic Soy Agar using PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., California, USA) following the manufacturer's instruction. These original DNA extracts were diluted by more than 20-fold to certain concentrations based on the measurements on NanoDrop ND-1000 (Thermo Scientific, Delaware, USA) to prepare the templates for qPCR.
The wheat head and stalk samples were returned to the lab within 1 hr of sampling and stored at −80° C. The grain samples were stored at room temperature after being brought back from the field. These wheat samples were ground up in liquid nitrogen immediately prior to DNA extraction. The composite residue samples were stored at 10° C. after being brought back to the lab and then DNA extraction was done within 24 h directly from the samples without grinding them up. Total DNA was extracted from approximately 0.3 gram of each sample using PowerSoil™ DNA Isolation Kit following the manufacturer's instruction, yielding 100 μl DNA extract per sample. Since the DNA concentrations of the wheat head and grain samples were so high (due to the abundant wheat DNA from fresh tissue) as to interfere with the interpretation of qPCR data, 1:20 dilutions of the original DNA extracts were used as templates for all those samples. Non-diluted DNA extracts were used as the templates of postharvest residue samples from Field One.
Quantitative PCR (qPCR). qPCR was performed as described in Example 1.
Culture-based quantification. Grain samples from Field Two at 155 days postharvest were quantified for 3C population using a dilution plating method. 1±0.01 g grains from each of the 12 bags (respectively for 12 plots, 6 inoculated and 6 non-inoculated) were transferred using a sterile spoon into a sterile polypropylene tube with a capacity of 15 ml. 2 ml sterile aqueous solution containing 10 mM K2HPO4, 10 mM KH2PO4 and 0.04% Tween 80 (Sigma-Aldrich, Missouri, USA) was added to each tube. The tubes were vortexed for 20 sec, shaken at room temperature at 200 revolution/min for 10 min and then vortexed again for 20 sec. 200 μl of resulting suspensions and their 10-fold and 100-fold dilutions was spread on each plate of ⅕-strength Tryptic Soy Agar amended with 1 ppm Prosaro® fungicide (Bayer CropScience LP, North Carolina, USA), 50 ppm Streptomycin, 50 ppm Kanamycin and 50 ppm Rifampicin (Sigma-Aldrich). 3C populations were enumerated as colony forming units (CFU) of yeast-like morphology on the plates after 5 days of incubation at 25° C.
Data processing and analysis. The standard series of 1, 1×10−1, 1×10−2 . . . 1×10−7 ng/μl 3C genomic DNA in template were run in triplicate to obtain the regression equation Y=5.792−0.2984X. This equation was used to calculate from the field samples' threshold cycle values (X) their log starting quantity values (Y) as equivalences to 3C genomic DNA concentration in template. The unit of starting quantity was converted from “ng/μl 3C genomic DNA in template” to “target gene copy number per gram sample. Unit conversion was based on the following two assumptions: (i) there was one copy of target gene per genome, supported by BLAST search of this gene and the h31.2 primers against the genome of 3C (accession numbers CAUG01000001-CAUG01000712) and by the sequencing coverage of the respective contig relative to the coverage of the whole genome; (ii) the genome size of 3C was about 0.02 pg per haploid genome (estimated based on the genome sizes of Cryptococcus gattii and C. neoformans) (ref. Kullman, B., Tamm, H. & Kullman, K. 2005. Fungal Genome Size Database. http://www.zbi.ee/fungal-genomesize). Accordingly, 1 ng 3C genomic DNA was considered equal to 5×104 copies of target gene and, reciprocally, one copy of target gene equal to 2×10−5 ng 3C genomic DNA. In this study, each reaction started with 2.5 μl template, which belonged to a total of 100 μl DNA extract resulted from 0.3 gram sample. Therefore, 1 ng/μ13C genomic DNA was considered equal to 3.3×108 (108.5) copies of target gene per gram sample for the wheat head and grain samples (1:20 dilutions of original DNA extracts as templates) and 1.7×107 (107.2) copies per gram sample for postharvest residue samples from Field One (non-diluted DNA extracts as templates).
The theoretical limit of detection for any given PCR reaction was considered to be one copy of target gene per reaction. Given the two aforementioned assumptions for unit conversion and that each reaction started with 2.5 μl template in this study, the theoretical detection limit of this study was 2×10−5 ng 3C genomic DNA per 2.5 μl, equal to 8×10−6 ng/μ13C genomic DNA in template. For the field samples, of which a total of 100 μl DNA extract resulted from 0.3 gram sample, the theoretical detection limits were 2.7×103 (103.4) copies of target gene per gram wheat head tissue for the wheat head and grain samples and 1.3×102 (102.1) copies per gram sample for the postharvest residue samples from Field One.
In the analysis on qPCR data, only the samples where a single peak of 87.0 or 87.5° C. (melting temperature of the standard series) was the only peak recognized by the iQ™5 detection system were considered as positive for detection of 3C. The samples showing no peak of 87.0 or 87.5° C. were considered as no detection of 3C and their Threshold Cycle (Ct) values were considered as 41 for statistical analysis. The samples showing a peak of 87.0 or 87.5° C. as well as other peaks were trimmed out of further analysis since they showed detection of 3C but the quantification will not be precise enough due to the interference of non-specific peaks. A few statistical outliers were also trimmed out. For each experiment, the trimmed-out data points never exceeded 7% of the total data points, unless specified. Analysis of variance using General Linear Model where α=0.05 was performed on the ranked forms of Ct values using the software Minitab 16 (Minitab Inc., Pennsylvania, USA).
Results
Dynamics of 3C Population in Artificially Inoculated Areas.
The population density of 3C on wheat heads, represented by the target gene copy number per gram wheat head tissue, was fluctuating over time in the areas artificially inoculated with 3C (
3C population density on majority of the samples was between 105.5 and 106.0 copies of target gene per gram wheat head tissue immediately post inoculation (0 DPI), decreased to between 104.5 and 105.5 one day later and then gradually increased to between 106.0 and 106.5 by the end of the season. Given that the inoculation rate was 106.3 to 106.8 cells per square centimeter and that the horizontal section of a wheat head was around one square centimeter, the 3C population density was maintained at a level that was lower than but still comparable to the inoculation rate in the field during the growing season.
Spread of 3C to the Non-Inoculated Area of Field.
There was a trend that the 3C population density decreased as the distance from inoculated area increases and that the 3C population density in non-inoculated area increased over time from 1 to 44 DPI (
There were a few samples in non-inoculated area on 0 DPI that were detected as 3.3×102 to 3.3×103 target gene copies per gram wheat head, while all the non-inoculated samples on 1 DPI showed no detection of 3C. However, this should not be considered as strong evidence that 3C population density was higher in non-inoculated area on 0 DPI than on 1 DPI, since those few 0-DPI samples were merely around the theoretical detection limit (2.7×103 target gene copies per gram wheat head).
The Effect of Sample Collection Activity on 3C Spreading.
To test if sample collectors walking throughout the field contributed to the spreading of 3C, additional wheat head samples were collected from borders of wheat-grown area in Field One on 10 DPI. Since sample collectors walked along the fallow walkways in between the wheat-grown area, it was possible that they brought 3C from artificially inoculated area to noninoculated area. It was hypothesized that if sample collection activity did contribute to 3C spreading, the wheat heads at the borders of wheat-grown area neighboring the walkways should show higher 3C population density than those deeper inside of wheat-grown area do. The samples relevant to this hypothesis were collected from the eastern side of the field (see
The Effect of Prevailing Wind Direction on 3C Spreading.
To test if the direction of prevailing wind (from west to east) affected the patterns of 3C spreading, the wheat head samples from the western and eastern sides of Field One were compared. The samples from different location groups (see
Overwintering Persistence of 3C.
To test if 3C can persist in the field over the winter, postharvest residues in the three inoculated areas of Field One were sampled in the winter to early spring of 2012. The data showed that 3C could still be detected in inoculated area by 6 to 9 months postharvest (
3C Population Level Around Harvest and During Storage
To examine the dynamics of 3C population on wheat around harvest, additional wheat head and grain samples were collected from another field (Field Two) at different time points around harvest. The 3C population levels on grains were examined again after a period of storage. The inoculated samples showed generally higher 3C detection than the non-inoculated samples for both sample types at different time points (
Culture-Based Quantification of 3C on Grains in Comparison to qPCR.
To investigate how well the DNA molecules detected by qPCR assay represent viable 3C cells, 3C population was quantified by colony forming units (CFU) on threshed grains at 155 days postharvest. Consistent with the qPCR results, CFU level of 3C was significantly higher on inoculated than on non-inoculated samples (
Filamentous fungi were also observed on the CFU quantification plates. The dominant colonies were identified by ITS regions as Alternaria alternata and Clasdosporium clasdosporioides, which are common saprophytes. The population levels were lower than 103 CFU per gram grain for both of the dominant filamentous fungi on most of the plates and did not greatly interfere with the quantification of 3C.
Discussion
Effectiveness of the qPCR Assay.
AqPCR assay of SYBR® Green chemistry was developed to monitor 3C dispersal and persistence on field-grown wheat and residues. This assay enabled the detection of 3C with clear differential between the wheat head samples supposedly positive versus negative for 3C colonization (
The sensitivity of the assay is good when compared to other related qPCR assays. Previous studies using qPCR to detect fungal pathogens on wheat showed minimum detections at the magnitude of 101 gene copies per reaction and 104 gene copies or 1 to 103 ng genomic DNA per gram wheat tissue, which were comparable to or higher than those in the assay (12.5 gene copies per reaction and 103 to 104 gene copies or 0.01 to 0.1 ng genomic DNA per gram wheat tissue) (
3C colonization of wheat heads has been previously quantified by colony forming units (CFU) (ref. Schisler 2009, 2010 Nat. FHB Forum Proc.). Applying 3C inoculum of approximately 3×108 CFU/ml at the rate of about 106 CFU per square centimeter resulted in 101 to 102 CFU/g glume tissue at 16 h post inoculation and 105 to 106 CFU at 256 to 280 h post inoculation. 3C CFU level decreased by a magnitude of 104 to 105 compared to the application rate in the 16 h post inoculation in their studies. In contrast, there was only an approximately 10-fold decrease in 3C DNA density during the one day post inoculation in this study. This dramatic difference is possibly due to that most of the 3C DNA detected on 1 DPI in this study belonged to non-viable cells or was already released from lysed cells. However, 3C CFU level increased to the similar level to the application rate ten days later in their studies, which is also the case in this study. This indicates that most of the 3C DNA detected on 10 DPI belonged to viable cells.
The disclosed assay showed a certain level of inter-strain specificity within the species of Cryptococcus flavescens, where six strains other than 3C were differentiated into two genotypic groups based on amplification efficiency (
3C Population Dynamics Across the Field
It is well known that microbial populations die off when they reach the death phase of growth. In some studies on the population dynamics of plant-associated bacteria using culture-based methods, decreased bacterial population on flowers or leaves of host plants later in the experimental time courses were observed. In a study using qPCR of Taqman chemistry for population quantification, the total population of three Fusarium species died off significantly on spring wheat stubble residue in the six months following summer harvest. In previous studies on 3C colonization of wheat using CFU-based quantification, slight dieoff of 3C population was observed between 184 and 256 to 280 h after wheat flowering and inoculation in the 2009 run but not in the 2010 run. In contrast, the disclosed study showed no observation of significant die-off of 3C population on the same types of samples over time, except on wheat heads from 0 to 1 DPI (
The dispersal pattern of 3C across the wheat field in this study resembles the dispersal of several fungal diseases on wheat. In these situations, at the same time point, the microorganism population decreased steeply from artificially inoculated area (source) to non-inoculated area (sink) near the source, while decreased gently over farther distances across the sink, especially at early time points. In this study, there were obvious increases in 3C population at the same distances in the sink over time (
Application of the qPCR Assay.
This assay can be used to conduct environmental fate and risk assessment of 3C as a biopesticide and to study the effects of common field abiotic and biotic factors of concern on 3C population dynamics in order to optimize the formulation of 3C-based biopesticides. It can also be used to investigate the association between 3C population dynamics and biocontrol efficacy of 3C against Fusarium head blight under certain microenvironments. This will facilitate the development of 3C as a biopesticide and provide guidance in the frequency and timing of 3C application. And the study on the correlation between the qPCR- and culture-based population quantification of 3C will enable better interpretation of the qPCR assay results. In addition, the target genes of this assay and other conserved genes of multiple C. flavescens strains can be sequenced to develop related qPCR assays that have different levels of specificity to serve different research purposes.
Materials and Methods
Strains. Cryptococcus flavescens strain OH182.9—3C (3C) was obtained from the ARS Culture Collection at the NCAUR laboratory under accession number NRRL Y-50378. Twelve other C. flavescens strains were also obtained from the ARS Culture Collection under accession numbers Y-1401T (=CBS 942T), Y-7372, Y-7373, Y-7374, Y-7375, Y-7376, Y-7377, Y-7379, YB-328, YB-601, YB-602 and YB-744. The information on strain isolation is summarized in Table 4. All the strains were maintained as glycerol stocks at −80° C. for long term storage.
Identification of MLST loci and choice of primers. The target genes of MLST were identified from 3C draft genome (GenBank accession numbers CAUG01000001 to CAUG01000712) through blastn and blastx search against NCBI Nucleotide collection (nr/nt) database. The boundaries of exons and introns were defined through Augustus prediction and alignment to C. neoformans JEC21 (GenBank accession numbers AE107341 to AE107356). Primers used for MLST were designed using software Primer 3 (Table 5).
DNA isolation, PCR and sequencing. Genomic DNA of 3C and the 12 other C. flavescens strains were extracted from 2-day old cultures grown on ⅕ Tryptic Soy Agar using the PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., California, USA) following the manufacturer's instruction. Each PCR reaction had a total volume of 25 μl, consisting of 2.5 μl genomic DNA of a C. flavescens strain, 0.2 μl solutions of each primer (100 μM), 2.5 μl mixture of four deoxynucleoside triphosphates (2 mM each, mixed at 1:1:1:1 ratio), 1.8 μl 25 mM MgCl2, 0.3 μl GoTaq® DNA polymerase (5 U/μl), 5 μl 5× Colorless GoTaq® Flex Buffer (Promega Corporation, Wisconsin, USA) and DNA-free PCR water as the rest of the volume. Thermal cycling program of 95° C. for 7 min, then 33 cycles of 94° C. for 1 min and 72° C. for 2 min, followed by 72° C. for 8 min was run on Peltier Thermal Cycler PTC-200 (MJ Research, Inc., Massachusetts, USA). Both strands of amplification products were sequenced by the Molecular and Cellular Imaging Center at The Ohio State University (Wooster, Ohio, USA). Sequences were edited and concatenated using software Geneious 6.1.5 (created by Biomatters). The majority of processed sequences were in high quality, where greater than 95% of the bases had Phred quality scores >40. The few lower-quality bases were manually examined and base-called with enough confidence that they did not compromise the accuracy of phylogenetic analysis.
C.
flavescens strains used in Example 4
aThis information is from the “Source” field of NRRL catalog and may not reflect the actual geographic location of isolation, which could not be identified from available records.
Phylogenetic analysis. Processed sequences were aligned and sequence identities were calculated using ClustalW2. Phylogenetic trees were generated using Maximum Likelihood method with General Time Reversible model in MEGA 5. Bootstrap test of 1,000 replicates were conducted to determine the reliability of consensus trees. To evaluate the effect of using different tree building methods, trees were also generated for all the relevant alignments using Neighbor Joining method in MEGA 5 and Bayesian inference in MrBayes v3.2.0, resulting in equivalent tree topologies to those by Maximum Likelihood method.
Biochemical assay. Profiles of carbon source utilization and chemical sensitivity were generated using Biolog GEN III Microplates (BIOLOG, Inc., California, USA). Two-day-old C. flavescens colonies grown on ⅕ Tryptic Soy Agar were suspended in 120 μl of sterile double distilled water. Optical density of cell suspensions was measured at 595 nm using an ELx800 Universal Microplate Reader (BIO-TEK Instrument, Inc., Vermont, USA). Forty to 100 μl of cell suspension in water was added to 10 ml inoculation fluid IF-A (BIOLOG, Inc., California, USA) and mixed well by vortexing. One hundred μl of cells suspended in IF-A (3×105 to 4×105 cells) was added to each well of a Biolog Gen III Microplate. Assay plates were incubated at room temperature (23 to 25° C.) in the dark. Color change of assay wells, representing C. flavescens growth and cell respiration, was measured as OD595 by the ELx800 Universal Microplate Reader following incubation. Two experiments were set up. Twelve strains (3C, Y-7372, Y-7373, Y-7374, Y-7375, Y-7376, Y-7377, Y-7379, YB-328, YB-601, YB-602 and YB-744) were used in Experiment One, and four strains (Y-1401T, 3C, Y-7372 and YB-601) were used in Experiment Two.
Statistical analyses were conducted on OD595 data in Minitab 15 (Minitab Inc., Pennsylvania, USA). Cluster Variables analysis was performed on the data from all the assay wells as a whole using Correlation Coefficient Distance and a variety of Linkage methods including Average, Complete, McQuitty, Single and Ward. To avoid mistaking run-to-run variation for real phenotypic difference among strains, the minimum pair-wise similarity percentage between the two independent runs of the same strain was used as the lower similarity threshold among the strains forming one distinct group. Analysis of Variance (ANOVA) was performed on the data of individual assay wells to compare distinct groups using General Linear Model with an α value of 0.01, 0.03 or 0.1 for defining significant difference in F test. In Experiment One, the data from 7 days of incubation in Run I and 10 days in Run II (referred as short incubation) were combined and those from 12 days in Run I and 15 days in Run II (referred as long incubation) combined for ANOVA. In Experiment Two, the data from the same days of incubation in both runs were combined for ANOVA.
Biocontrol assays. Macroconidia of F. graminearum isolate Z-3639 were produced on clarified V8 juice agar (CV8 agar) under a regime of 12 h/day fluorescent light for seven days at 25° C. Colonized plates were flooded with weak PO4 buffer to obtain conidial suspensions of the pathogen. Biomass of all C. flavescens strains except Y-7374, Y-7375, Y-7376, Y-7377, Y-7379 was initiated on plates of one-fifth strength Tryptic soy broth agar (TSBA/5). After 24 h incubation at 28° C., cells were used to inoculate 50 ml of semi-defined complete liquid medium (SDCL) in 250-ml Erlenmeyer flasks (OD=0.10, A620). Flasks were incubated in a shaker incubator (250 rpm, 25° C., 24 h) to produce cells in log growth to inoculate cultures (50 ml SDCL in 250-ml flask, OD=0.10, A620) that were then used as inoculum cultures after a 48 h incubation.
Each experiment to determine strain efficacy was conducted in a climate-controlled greenhouse with temperatures that ranged from 17 to 20° C. at night and 25 to 28° C. during the day. Natural sunlight was supplemented with high-pressure sodium lights for 14 h/day. Each experimental unit consisted of two plants of hard red spring wheat cultivar Norm grown in a 19-cm-diameter plastic pot. Each pot contained air-steam pasteurized (60° C. for 30 min) potting mix (Terra-lite Redi-earth mix, W. R. Grace, Cambridge, Mass.) and plants were grown in a growth chamber (25° C., 14-h photoperiod, 600 u′ mol/[m2/s]) for 7-8 weeks prior to transfer to greenhouse benches. Pots were fertilized after 1 week and weekly thereafter with 50 ml of a solution containing 1.25 g/liter Peters 20-20-20 (Grace-Sierra Horticultural Products, Milpitas, Calif.) and 0.079 g/liter iron chelate (Sprint 330, Becker Underwood, Inc., Ames, Iowa). Approximately one week after transferring plants to the greenhouse, wheat heads were inoculated at anthesis by spraying with 25% freshly harvested C. flavescens cells (˜5×107 CFU/ml) in PO4 buffer and a final concentration of 0.036% Tween 80 (Sigma Chemical Co., St. Louis, Mo.). For each experiment, 50 mL of inoculum of each strain was used to inoculate six plants representing a total of 12 to 15 heads. Heads were then challenged immediately by spraying 12 mL of a conidial suspension of F. graminearum (3×104 conidia/ml) in PO4 buffer with 0.036% Tween 80. Wheat heads inoculated first with the PO4 buffer/tween 80 solution and then only with F. graminearum conidia in PO4 buffer/tween 80 served as a pathogen only control. Uninoculated pots of wheat were used to ensure pathogen inoculum did not spread between treatments. Inoculated plants were misted lightly with distilled water and incubated in a plastic humidity chamber at 17 to 20° C. at night and 25 to 28° C. during the day for 3 days before being transferred to greenhouse benches. Treatments were arranged in a completely randomized design. Fusarium head blight severity was visually estimated using a 0 to 100% scale at 10 and 16 days after inoculation. All greenhouse experiments were conducted five times. Data were analyzed using the Kruskal-Wallis test using Minitab v.15.
Accession numbers. The sequences used in MLST analysis were deposited in the GenBank/EMBL/DDBJ databases under accession numbers KC679238 to KC679297 and KF171106 to KF171122.
Results
Evidence for Two Distinct Genotypes of C. flavescens.
Six loci-rDNA-ITS, the partial sequences of four protein-encoding genes and cs22 were sequenced and aligned to infer phylogenetic relationship among the C. flavescens strains (Table 5). These loci were probably unlinked since they were from six different scaffolds in the draft genome of 3C and their homologs were located on six different chromosomes of C. neoformans JEC21 (GenBank accession numbers AE107341 to AE107356), respectively. The rDNA-ITS sequences of all 13 strains were 100% identical. The other five loci revealed divergence within the species (thus termed variable loci), resulting in two clades (genotypes A and B) supported by a bootstrap value of 100 (
The genotype split was consistent across all the five variable loci (
Taxometric Analysis of Biochemical Properties of C. flavescens Strains.
While sharing many similarities in response to the Biolog GEN III substrates, 12 C. flavescens strains (not including Y-1401T) could be split into three measurably different groups, depending on incubation time (
aThe percentage of columns in the alignment for which all the sequences were identical.
bThe strains classified as genotype A are 3C, Y-1401T, Y-7373, Y-7377, YB-601 and YB-602; those classified as genotype B are Y-7372, Y-7374, Y-7375, Y-7376, Y-7379, YB-328 and YB-744.
Genotype-A strains showed different subgrouping patterns depending on incubation time (
aInformation in these rows is based on the Experiment One data combined from 12 days of incubation in Run I and 15 days of incubation in Run II. Y-1401T was not included.
bInformation in this row is based on the Experiment Two data combined from 19 days of incubation in both Run I and Run II.
In subsequent analyses, Type strain Y-1401T showed distinct biochemical properties from 3C, YB-601 (both genotype A) and Y-7372 (genotype B) regardless of incubation time (data not shown). This distinction was probably due to the poorer growth of Y-1401T than the other three strains, with statistical significance in 27 out of 71 carbon source utilization assays and 16 out of 23 chemical sensitivity assays (Table 7, Row 4). The only exception was that Y-1401T utilized citric acid slightly better than the other strains by the end of 19 days' incubation (P=0.086 for F test). For each individual run, 3C and Y-7372 formed a different cluster from YB-601 after 5 days' incubation, while 3C and YB-601 formed a different cluster from Y-7372 after 19 days' incubation. These patterns were consistent with the two runs (
Biocontrol Efficacy of the Two C. flavescens Genotypes in Suppressing Fusarium Head Blight (FHB).
Representative strains from the two genotypes were assessed for FHB suppression on wheat in five separate greenhouse bioassays. Reductions in FHB disease severity by pre-inoculation with C. flavescens isolates were observed in four of five independent trials. In two of those four trials, the observed reduction in disease was statistically significant (P<0.10, Kruskal-Wallis test) after 16 days of incubation. In total, the genotype-A isolates displayed greater biocontrol activity than the genotype-B isolates in three of five independent trials, but given the high degree of variation of disease scores that are typical of such trials, the individual run results were not statistically significant. However, considering all of the tested strains, significant reductions in disease severity due to inoculation with C. flavescens were observed at both 10 and 16 days post-inoculation (P=0.11 and 0.03, respectively, Kruskal-Wallis test;
Discussion
This example provides the first detailed characterization of C. flavescens diversity, and demonstrates the occurrence of genotypically and phenotypically distinct subgroups within the large majority of publicly available isolates of the species. Previously, the only available sequences of C. flavescens were the ribosomal DNAs and internal transcribed spacers of type strain Y-1401T (=CBS 942T) and several other strains. In contrast to the intra-species diversity at the rDNA-ITS loci of human-pathogenic C. neoformans and biocontrol fungus Trichoderma harzianum, all of C. flavescens rDNA-ITS sequences obtained in this study were 100% identical to the published sequence of Y-1401T (GenBank accession number AB035046). Since a strain with 99.0% identity to Y-1401T in ITS region has been identified as C. flavescens, all the other 12 strains in this study would typically be classified as C. flavescens as well based on ITS genotype alone. However, this group was capable of being split into two distinct genotypes based on MLST (
The genotype split shown by the five variable MLST loci was consistent with the results observed during the development of a quantitative PCR (qPCR) assay for C. flavescens (see Example 5). In Example 5, a 150-bp region within the same heat shock protein 70 kDa locus used in MLST of this study was targeted. The qPCR assay differentiated the tested C. flavescens strains into two groups, 3C like and non-3C like, based on difference in threshold cycles that resulted from sequence difference in primer binding regions. The 3C like strains actually belong to genotype A and non-3C like to genotype B. Within genotype A, the 100% identity of Y-1401T to 3C and Y-7373 at the five variable MLST loci indicates that Y-1401T, though isolated in Japan many decades earlier, is closely related to the two strains isolated more recently in the USA. Thus, the species appears to be genetically stable on a decadal time scale.
The utilization results of carbon sources were consistent most of the time between previous studies and this study (Table 7, Row 5). The only minor inconsistency was glycerol. The results were previously negative for Y-1401T or variable. However, it was positive or near positive for utilization by all the 13 strains in the present study (OD595=0.589 for Y-1401T and OD595>0.7 for the other 12 strains after longest incubation). Though Y-1401T showed minimal genetic divergence from most of the genotype-A strains, its Biolog assay results were highly distinct due to the generally poorer growth. It is unknown whether this pattern resulted from geographic/habitat difference or sub-culturing/storage of strains over the years. Carbon source utilization has been previously related to recovery of biocontrol agents against Fusarium head blight (FHB), where utilization of tartaric acid by microorganisms was positively biased towards the ability to reduce FHB damages. Tartaric acid can be utilized by OH182.9 (progenitor of 3C) but not included in the Biolog assays used in this study.
Several strains were previously designated as two mating types based on a mating test where mating responses included formation of conjugation tubes and hypha growth from fused tubes in mixed culture of strain pairs. The strains of the opposite mating types are able to mate (in the lab) while those of the same mating type are not. In that study, Y-7372, Y-7374, Y-7375, Y-7376 and Y-7379 were designated as mating type α; while Y-7373 and Y-7377 as mating type a. All the five α-strains were shown as genotype B while both a-strains as genotype A by MLST in the present study. The genetic divergence between the two mating types indicated minimal sexual recombination among the studied strains in the environment. The fact that the strains of the same MLST genotype belonged to the same mating type in this study is consistent with a previous MLST study on C. neoformans var. grubii, where more than 40 MLST types and only two mating types were identified. Thus, within a Cryptococcus species, the strains of different mating types tend to be of different genotypes at other conserved loci.
Genetic markers have been used to assist discovery of novel biocontrol isolates and to study the geographic distribution of biocontrol agents. The MLST markers developed in this study may be used to identify new and novel C. flavescens biocontrol agents. MLST analysis can also be performed on additional C. flavescens strains when they are recovered from wheat fields at various geographic locations to comprehensively characterize the natural range and diversity of this species. Heterosis, where hybrid progeny shows superior biological quality over parents, has been utilized in plant breeding to produce hybrid cultivars with improved performance such as higher yield and disease resistance. Crossing C. flavescens isolates of compatible mating types can be conducted to investigate whether hybrid progeny shows increased biocontrol efficacy against FHB. The carbon source utilization and chemical resistance patterns shown in Biolog plates may also be used to develop C. flavescens-specific media aiding the selective recovery of C. flavescens as potential biocontrol agents.
Materials and Methods
Strains. Cryptococcus flavescens strain OH182.9—3C (3C) was obtained from the ARS Culture Collection at the NCAUR laboratory under accession number NRRL Y-50378. Six other C. flavescens strains were also obtained from NRRL under accession numbers Y-7372, Y-7373, YB-328, YB-601, YB-602 and YB-744. All the strains were maintained as glycerol stocks at −80° C. for long term storage.
Field establishment and 3C application. The experiments of this study were performed in two fields planted with soft red winter wheat (Triticum aestivum L.) in the Snyder Farm at the northern end of Wooster, Ohio. The first field, subsequently noted as Field One, was planted with Cultivar Hopewell on Oct. 11, 2010. The layout of Field One is shown in
Inoculum production. Log-phase cells of 3C produced in SDCL medium served as a 5% seed inoculum for the production in B Braun D-100 fermentor charged with 80 liters of SDCL medium. The fermentor was operated at 25° C., 20 liter/min aeration and 200 revolution/min with agitation provided by twin Rushton impellers. Eighteen hours after inoculation, fermentor temperature was reduced to 15° C. to cold shock the cells for 24 h prior to harvest. The cells were harvested through concentration into a paste using a Sharples 12-V tubular bowl centrifuge. The cell paste was then suspended in weak PO4 buffer at approximately 2×109 CFU/ml and frozen at −20° C. as stock. The field inoculum was prepared right before application by mixing 1.5 liter of stock (pre-thawed at 10° C. overnight) with 1.5 liter sterile double distilled water and 10.8 ml 10% Tween 80. During anthesis of the wheat (Feekes Stage 10.5, on May 29, 2011), three areas of 6.1 m by 6.1 m (Plots i—1, i—2, and i—3 of
For producing 3C inoculum for Field Two, 500-ml flasks containing 200 ml SDCL medium inoculated with 3C (starting OD of 0.10 at A620) were incubated at 25° C. at 250 revolution/min for 24 h. These cultures were then used to seed 3-liter Fernbach flasks containing 1.25 liter of SDCL medium to an OD of 0.10. Cultures were incubated for 48 h at 25° C. and 250 revolution/min. Colonized broth of 2.3×108 CFU/ml was then transferred into sterile containers, transported to the field on ice and applied at the rate of 4.4×105 CFU per square centimeter in the field within 24 h. The application was performed during anthesis of the wheat (Feekes Stage 10.5, on May 29, 2012). Sampling for this study was performed in 12 plots, 6 of which were sprayed with 3C only and another 6 of which were not sprayed with any microorganism. The plot locations of different treatments were randomized across the field.
Sample collection. Wheat head samples were collected by clipping at the stalk 1 to 2 cm below the head and sealing each head in an individual sample bag. During sample collection, each head was only touched with the inside of the bag used to collect it and the clipping scissors were sterilized with 75% aqueous ethanol between clips to prevent cross-contamination. Heads were collected from Field One within 1 h after the spray application of 3C (0 day post inoculation) as well as 24 h (1 day), 10, 26 and 44 days post inoculation in the summer of 2011 (see
Postharvest residue samples were collected from the three inoculated areas of Field One in late January and late March of 2012. Seven different locations were sampled within a circle of about 1-m radius around the center of each inoculated area. At each time point, two types of samples were collected from each location: a mixture of soil, living weeds and brownish rotting plant materials was collected as one composite sample, and non-rotting wheat stalks were collected as the other sample.
The grains were harvested from each plot as a whole for the 12 plots of Field Two on Jun. 26, 2012. The total grains of each plot were stored in one paper bag as a layer thinner than 6 cm at dark at room temperature. One subsample of approximately 10 g was collected from the total grains of each plot immediately prior to grinding up for DNA extraction at 14, 64 and 155 days postharvest, respectively.
Sample handling, DNA extraction and template preparation. Genomic DNA of 3C and the six other C. flavescens strains was extracted from 2-day old cultures grown on ⅕ Tryptic Soy Agar using PowerSoil™ DNA Isolation Kit (MO BIO Laboratories, Inc., California, USA) following the manufacturer's instruction. These original DNA extracts were diluted by more than 20-fold to certain concentrations based on the measurements on NanoDrop ND-1000 (Thermo Scientific, Delaware, USA) to prepare the templates for qPCR.
The wheat head and stalk samples were returned to the lab within 1 hr of sampling and stored at −80° C. The wheat (including grain) samples were ground up in liquid nitrogen immediately prior to DNA extraction. The composite residue samples were stored at 10° C. after returned to the lab and then DNA extraction was done within 24 h directly from the samples without grinding in liquid nitrogen. Total DNA was extracted from approximately 0.3 gram of each sample using PowerSoil™ DNA Isolation Kit following the manufacturer's instruction, yielding 100 μl DNA extract per sample. Since the DNA concentrations of the wheat head and grain samples were so high (due to the abundant wheat DNA from fresh tissue) as to interfere with the interpretation of qPCR data, 1:20 dilutions and 1:10 dilutions of the original DNA extracts were used as templates for those samples from Field One and Two, respectively. Non-diluted DNA extracts were used as the templates of postharvest residue samples from Field One.
Quantitative PCR (qPCR). qPCR primers were designed using Primer3 software based on the draft genomic sequences of 3C (EMBL accession number CAUG01000001 to CAUG01000712). qPCR test for specificity was performed on 14 pairs of primers targeting 7 putative conserved regions, using the 1 ng/μl genomic DNA of 3C and the other 6 C. flavescens strains as template. The 7 regions were putatively ITS1-5.8S-ITS2 and partial regions of the following protein encoding genes: (3-tubulin, chitin synthase 5, elongation factor 1 and three 70 kDa heat shock proteins. Among the 14 pairs of primers, Pair h31.2 showed the greatest difference in Threshold Cycle values between 3C and any of Y-7372, YB-328 and YB-744, and was thus chosen for use in the qPCR assay described as follows. Each qPCR reaction had a total volume of 25 μl, consisting of 2.5 μl template DNA, 0.4 pmol/μl of each primer (forward primer h31.2_F: 5′-CGTCAGCGTGTTGCCACTTCGT-3′ (SEQ ID NO: 67); reverse primer h31.2R: 5′-GCTGCTGTCTTGCGGTCGCTTA-3′(SEQ ID NO: 68)), 12.5 μl iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc., California, USA) and DNA-free PCR water as the rest of the volume. DNA-free PCR water was also used as the template of negative controls. The thermal cycling program was run on the iCycler and the fluorescence data was collected by the iQ™5 detection system (Bio-Rad Laboratories, Inc.). Amplification was two-step with 3 min at 95° C. followed by 40 cycles of 15 s at 95° C. and 1 min at 72° C. Then the melting curve was run from 55° C. to 95° C. with an increment of 0.5° C. and a dwelling time of 10 s at each temperature. The fluorescence data of qPCR was analyzed using the iQ™5 Optical System Software (Bio-Rad Laboratories, Inc.).
Data processing and analysis. The standard series of 1, 1×10−1, 1×10−2 . . . 1×10−7 ng/μl 3C genomic DNA in template were run in triplicate to obtain the regression equation Y=5.792−0.2984X (R2=0.991). This equation was used to calculate from the field samples' Threshold Cycle values (X) their log starting quantity values (Y) as equivalences to 3C genomic DNA concentration in template. The unit of starting quantity was converted from “ng/μl 3C genomic DNA in template” to “target gene copy number per gram sample”. This unit conversion was based on the following two assumptions: (i) there was one copy of target gene per genome, supported by BLAST search of this gene and the h31.2 primers against the draft genome of 3C and by the sequencing coverage of the respective scaffold relative to the coverage of the whole genome (data not shown); (ii) the genome size of 3C was about 0.02 pg per haploid genome (estimated based on the genome sizes of Cryptococcus gattii and C. neoformans) (Fungal Genome Size Database). Accordingly, 1 ng 3C genomic DNA was considered equal to 5×104 copies of target gene and, reciprocally, one copy of target gene equal to 2×10−5 ng 3C genomic DNA. In this study, each reaction started with 2.5 μl template, which belonged to a total of 100 μl DNA extract resulted from 0.3 gram sample. Therefore, 1 ng/μl 3C genomic DNA was considered equal to 3.3×108 (108.5) or 1.7×108 (108.2) copies of target gene per gram sample for the wheat head or grain samples (1:20 or 1:10 dilutions of original DNA extracts as templates, respectively) and 1.7×107 (107.2) copies per gram sample for postharvest residue samples from Field One (non-diluted DNA extracts as templates).
The theoretical limit of detection for any given PCR reaction was considered to be one copy of target gene per reaction. Given the two aforementioned assumptions for unit conversion and that each reaction started with 2.5 μl template in this study, the theoretical detection limit of this study was 2×10−5 ng 3C genomic DNA per 2.5 μl, equal to 8×10−6 ng/μl 3C genomic DNA in template. For the field samples, of which a total of 100 μl DNA extract resulted from 0.3 gram sample, the theoretical detection limits were 2.7×103 (103.4) or 1.3×103 (103.1) copies of target gene per gram sample for the wheat head or grain samples (1:20 or 1:10 dilutions of original DNA extracts as templates, respectively) and 1.3×102 (102.1) copies per gram sample for the postharvest residue samples from Field One.
In the melting curve analysis for qPCR, a single peak of 87.0 or 87.5° C. was the only peak reported by the iQ™5 detection system for the concentrated templates in the standard series (1 through 10−4 ng/μl 3C genomic DNA). Therefore for field samples, only those where a single peak of 87.0 or 87.5° C. was the only reported peak were considered as positive for detection of 3C. The samples showing no peak of 87.0 or 87.5° C. were considered as no detection of 3C and their Ct values were considered as 41 for statistical analysis. The samples showing a peak of 87.0 or 87.5° C. as well as other peaks were trimmed out of further analysis since they showed detection of 3C but the quantification will not be precise enough due to the interference of non-specific peaks. A few statistical outliers were also trimmed out. For each experiment, the trimmed-out data points never exceeded 7% of the total data points, unless specified. Analysis of variance using General Linear Model where α=0.05 was performed on the ranked forms of Ct values using the software Minitab 16 (Minitab Inc., Pennsylvania, USA).
Results
Sensitivity and Specificity of the Assay.
The primer pair h31.2 used in the qPCR assay were designed to amplify part of a putative heat shock protein 70 kDa (hsp70) gene identified from the draft genomic sequence of 3C. Primer h31.2F binds to a putative intron region and primer h31.2R binds to a putative exon region (
To test the specificity of this assay within the Cryptococcus flavescens species, qPCR was also run on the 1 ng/μl genomic DNA templates of six other C. flavescens strains than 3C (Table 8). The sequences of strains Y-7372, YB-328 and YB-744 differ from 3C by two base pairs at each primer binding site (
aThreshold Cycle was not detected (n.d.) by the software when fluorescence signal was below the threshold at the end of 40-cycle amplification.
bMelting temperature was not applicable (n.a.) for the samples for which the detection software reported no peak of 87.0 or 87.5° C. (the only reported peak for standard series 1 through 10−4 ng/μl in template).
cThe templates of the field samples are the 1:20 diluted DNA extracts from a 3C-inoculated wheat head 44 days post inoculation and from any non-inoculated head at a distance of 25.8 m to the nearest inoculated area on the day of inoculation.
Dynamics of 3C-Like C. flavescens Population in Areas Inoculated with 3C.
The population density of 3C-like C. flavescens on wheat heads, represented by the target gene copy number per gram wheat head tissue, fluctuated over time in the areas inoculated with 3C (
3C-like C. flavescens population on the majority of the samples was between 105.5 and 106.0 copies of target gene per gram wheat head tissue immediately post inoculation (0 DPI) and was between 104.5 and 105.5 on 1 DPI. Subsequently, population increased to between 106.0 and 106.5 by the end of the season. Given that the inoculation rate was 106.3 to 106.8 3C cells per square centimeter and that the horizontal section of a wheat head was around one square centimeter, qPCR-detected population was maintained at a level that was slightly lower than, but still comparable to, the inoculation rate throughout the growing season.
Dispersal of 3C to the Non-Inoculated Area of Field.
There was a trend that 3C-like population decreased as the distance from inoculated area increased, displayed by the percentages of samples above a threshold (3.3×104 gene copy per gram wheat head) around the minimal population density necessary to show biocontrol efficacy (
There were a few samples (i.e. 5 of 36) positive for 3C-like C. flavescens in the non-inoculated area at 0 DPI. However the level detected was 3.3×102 to 3.3×103 target gene copies of the target per gram wheat head, an amount around the detection limit of the assay. And, none of the non-inoculated samples at 1 DPI (i.e. 0 of 35) scored positive for 3C-like populations. These data indicate that colonization of wheat heads by native C. flavescens was generally infrequent and, when it did occur, never reached 104 gene copies per wheat head. Thus, the majority of qPCR-detected populations in this experiment were interpreted as arising from 3C inoculation, and this is why the aforementioned population dynamics over distances and time reflect the dispersal pattern of 3C.
The Effect of Sample Collection Activity on 3C Dispersal.
To test whether sample collectors walking throughout the field contributed to the apparent dispersal of 3C, additional wheat head samples were collected from borders of wheat-grown area in Field One on 10 DPI. Since sample collectors walked along the fallow walkways in between the wheat-grown area, it was possible that they brought 3C from inoculated area to non-inoculated area. It was hypothesized that if sample collection activity did contribute to 3C dispersal, the wheat heads at the borders of wheat-grown area neighboring the walkways should show higher 3C population density than those deeper inside of wheat-grown area do. The samples relevant to this hypothesis were collected from the eastern side of the field (see
The Effect of Prevailing Wind Direction on 3C Dispersal.
To test whether the direction of prevailing wind (from west to east) affected the apparent patterns of 3C dispersal, the wheat head samples from the western and eastern sides of Field One were compared. The samples from different location groups (see
Overwintering Persistence of 3C-Like C. flavescens.
To test whether 3C-like C. flavescens can persist in the field over the winter, the three inoculated areas of Field One were sampled in the winter to early spring of 2012. The data showed that population could still be detected in inoculated area by 6 to 9 months postharvest (
Population Levels of 3C-Like C. flavescens Around Harvest and During Storage.
Additional wheat head and grain samples were collected from another field (Field Two) at different time points around harvest. Population levels on grains were repetitively measured by qPCR after periods of storage. The inoculated samples showed generally higher population than the non-inoculated samples for both sample types at different time points (
Discussion
This example describes the development of a qPCR assay to monitor the dispersal and persistence of 3C-like Crypotococcus flavescens on field-grown wheat and, more broadly, in the environment. This assay enabled the detection with clear differential between the inoculated and non-inoculated wheat head samples (Table 8, last two rows). And it can be reasonably applied to multiple sample types, since it gave meaningful differential results within each of the sample types tested in this study. The sensitivity of the present assay was comparable to other related qPCR assays. Previous studies using qPCR to detect fungal pathogens on wheat showed minimum detections at the magnitude of 101 gene copies per reaction and 104 gene copies or 1 to 103 ng genomic DNA per gram wheat tissue, which were comparable to those in the present assay (12.5 gene copies per reaction and 103 to 104 gene copies or 0.01 to 0.1 ng genomic DNA per gram wheat tissue) (Table 8,
The qPCR assay showed intra-species specificity to four 3C-like C. flavescens strains (3C, Y-7373, YB-601 and YB-602) over the other three strains (Table 8). No further discrimination by Threshold Cycle (Ct) value nor melting temperature can be achieved among 3C-like strains, which is due to the high sequence similarity among them. Sequence difference between 3C and the three 3C-like strains was actually 0 to 1 bp across a total of more than 5500 bp of DNA (including ˜2000 bp putatively non-coding sequences) across 9 conserved regions, including all the 7 regions used for primer design in this study. Since mismatch by one base pair at primer binding site only gave rise to a Ct difference of ˜1.5 for one of the tested primers, it is almost impossible to differentiate among 3C-like C. flavescens through amplification from those conserved regions. It may be possible to design primers targeting more variable regions to achieve “3C-only” specificity. However, such a strategy may sacrifice sensitivity because the higher natural mutation rates in variable regions may result in loss of ability to detect 3C progenies that have mutations in primer binding sites. Furthermore, the possibility cannot be excluded that some 3C-like strains may have originated from the same population as 3C in the near past and were named as different strains simply due to separate isolation events. The biocontrol efficacy against Fusarium head blight did not differ significantly among 3C and other 3C-like strains. Therefore, further refinement of the assay to gain additional specificity to the isolate 3C will not provide much practical value.
Previous studies demonstrate that applying 3C inoculum of approximately 3×108 CFU/ml at the rate of about 106 CFU per square centimeter resulted in 101 to 102 CFU/g glume tissue at 16 h post inoculation and 105 to 106 CFU at 256 to 280 h post inoculation. In those studies, strain CFU of strain 3C decreased by a magnitude of 104 to 105 compared to the application rate in the 16 h post inoculation. In contrast, there was only an approximately 10-fold decrease in 3C DNA population during the one day following inoculation in the present study (
Previously developed qPCR assays of other wheat-associated eukaryotes showed inter-species specificities. The present assay showed a certain level of inter-strain specificity within the species of Cryptococcus flavescens. Specifically, assays of six other isolates of C. flavescens indicated that the species encompassed two genotypic groups based on the amplification efficiency of standard amounts of DNA template (Table 8). Even though all 3C-like isolates showed similar amplification efficiency, detection of native populations of 3C's close relatives was minimal in this study. This is because no detection events were noted across nearly all the non-inoculated samples on 0 and 1 DPI while the inoculated samples all showed clear detection (
In some studies on the population dynamics of plant-associated bacteria using culture-based methods, decreased bacterial population on flowers or leaves of host plants later in the experimental time courses were observed. In a study using qPCR for population quantification, the total population of three Fusarium species died off significantly on spring wheat stubble residue in six months after the harvest in summer. In the previous studies on 3C colonization of wheat using CFU-based quantification, slight die-off of 3C population was observed between 184 and 256 to 280 h after wheat flowering and inoculation in the 2009 run but not in the 2010 run. The present study showed trends of population decrease on wheat heads from 0 to 1 DPI (
The dispersal pattern of 3C across the wheat field in this study resembles those reported previously for several fungal pathogens on wheat. In such studies, at each given time point, the microorganism population decreased steeply from artificially inoculated area (source) to non-inoculated area (sink) near the source, while decreased gently over farther distances across the sink, especially at early time points. In the present study, there were obvious increases in 3C population at the same distances in the sink over time (
In previous studies, qPCR assays for quantification of wheat pathogens have been used to evaluate disease severity and to monitor and predict disease development. The present qPCR assay can be used to determine environmental fate of 3C-like C. flavescens as biopesticides. Specifically, dispersal and persistence of 3C-like C. flavescens can be investigated throughout the wheat production system at various geographic locations over multiple growing seasons. It can also be used to study the effects of common field abiotic and biotic factors of concern on 3C population dynamics in order to optimize the formulation of 3C-based biopesticides. Further, it can be used to investigate the association between 3C population dynamics and biocontrol efficacy of 3C against Fusarium head blight under various conditions.
One or more strains of Cryptococcus flavescens, one or more microbial antagonists, a composition, or groups of primer pairs described herein may be provided in the form of a kit. In some embodiments, the kit provides one or more strains of C. flavescens, microbial antagonists, or compositions for application to the seed head of a plant, most preferably a cereal plant.
In other embodiments, the kit provides a group of primer pairs designed specifically to amplify β-tubulin, chitin synthase 1, EF1, and/or hsp70. The kits may also provide one or more containers filled with one or more necessary PCR reagents, including but not limited to dNTPs, reaction buffer, Taq polymerase, and RNAse-free water. Optionally associated with such container(s) is a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of the group of primer pairs and associated reagents, which notice reflects approval by the agency of manufacture, use or sale for research use.
The kits may include appropriate instructions for preparing supplied C. flavescens strains, microbial antagonists, and compositions for application. Instructions may further describe appropriate application techniques and concentrations of prepared C. flavescens strains, microbial antagonists, and compositions. When group of primer pairs are included in the kit, the kit may include appropriate instructions for preparing, executing, and analyzing qPCR to determine the genotype of a C. flavescens strain using the primer pairs included in the kit. The instructions may be in any suitable format, including, but not limited to, printed matter, videotape, computer readable disk, or optical disc.
While the invention has been described with reference to various and preferred embodiments, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the essential scope of the invention. In addition, many modifications may be made to adapt a particular situation or material to the teachings of the invention without departing from the essential scope thereof.
Therefore, it is intended that the invention not be limited to the particular embodiment disclosed herein contemplated for carrying out this invention, but that the invention will include all embodiments falling within the scope of the claims.
It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application claims priority to U.S. Provisional Application No. 61/787,458, filed on Mar. 15, 2013, the entire disclosure of which is expressly incorporated herein by reference for all purposes.
| Number | Date | Country | |
|---|---|---|---|
| 61787458 | Mar 2013 | US |
