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Physical identification techniques

Microscope Identification

P.humuli spores have a unique size and shape allowing easy identification when examining a field sample. The spore is on average 11 nm by 7 nm when viewed at a magnitude of 100X but sizes can vary in both length and width. However, the width of the spore will never drop below 5 nm. When the sample is stained with Aniline blue, the cytoplasm of the spore will turn a dark blue. No gram stain is required for a proper identification. There will also be a physical gap between the capsule and the cytoplasm which is present in every spore. The cytoplasm will not touch the outer capsule in P. humuli spores. A distinctive point will be present in the capsule at one end forming a tear drop. Some variation exists with this pathogen with some spores forming with two points, one on either end, or with a complete lack of one altogether. Positive identification relies on several key factors, the time the spore was gathered, its dimensions, and its proximity to other positively identified spores.

There is a known spike in spore concentrations in late afternoon starting around 3pm. This spike lasts until 8pm and will drop to almost nothing during the night. Identification of spores from samples gathered at night will not produce definitive results. Around 7 AM the spore concentration will start to rise again and will continue to increase till late afternoon.

When viewing a sample from a concentration spike It is important to identify a standard spore to be used for comparison to others on the slide. If the spore meets all the criteria, then it can be used as a standard and looked back at to determine whether a questionable spore is P. humuli.

Spore Trap Identification

P. humuli can be easily detected under a microscope because of its unique tear drop shape. The primary mechanism for collecting these spores is a machine called a spore trap which provides a snapshot of the local environment imprinted on cellophane tape [2]. A spore trap runs for a total of seven days running constantly, gathering Spores and other plant material. The material enters the trap through a small orifice and are imprinted onto a reel of Vaseline covered cellophane tape. The tape is attached to a clock motor that spins the tape at a constant pace allowing each hour to be recorded on a new section of tape. This seven-day cycle is used to identify trends in spore concentrations within a local county. Once the concentration rises above 10 spores a day fungicide trials can be scheduled and applied to the field to prevent an outbreak within the area.

Following this cycle, the tape is removed through an opening at the top and refrigerated. The tape is then laid out on a board that has 23 evenly spaced grooves. Using a razor blade these grooves are carefully imprinted onto the surface of the tape without cutting the tape itself. These lines separate each of the 24 hours allowing easy differentiation between morning and evening samples. Each 24-hour segment of tape is then covered in Crystalline blue dye and a cover slide is sealed to the surface. The slide can then be examined immediately under the microscope. The hour lines make recording positive spore identification more efficient.

Genetic Identification Techniques

Current methods of early detection, spore trap microscopy, do not sufficiently identify individual species of Downy Mildew as their Sporangium morphologies are identical. To determine the species of Downy Mildew two assays have been develops utilizing PCR and qPCR(real time PCR). Both assays require purified DNA from the pathogen extracted from infected rhizomes, foliage, or the stem [3]. A common extraction process used to isolate P. humuli is the CTAB assay (cetyl trimethylammonium bromide), which takes a total of two days and produces an average of 35ul of DNA solution. The concentration of the final product is unknown until measured on a Nano dropper or qBit machine. CTAB works more efficiently with samples from the leaves because the concentration of sporangia is high [3]. There is also less microbiome diversity on the surface of the leaf reducing the probability of contamination from bacteria and fungi. In root rhizomes the Diversity of the Microbiome is much higher increasing the possibility for contamination. At the same time the spore concentration is low making DNA amplification more difficult. The rhizome is required for Hop propagation and serves as a natural reservoir for P. humuli. Early diagnosis is crucial to reducing disease transmission and an assay for testing the rhizome is in the early stages of development.

PCR Identification

Current methods of DNA extraction use PCR, Polymerase Chain Reaction, to provide positive identification of P. humuli spores at low concentrations [1]. Field soils can now be tested, a reservoir of the pathogen, allowing new acres of farmland to be declared clean before planting. Using the CTAB DNA extraction assay, P. humuli DNA can be extracted from samples collected in both the Spore trap and the field [3]. This allows spore samples to be morphologically identified as a species of Downy Mildew and then put through the DNA isolation process. Once a sample has been purified, a PCR assay is constructed using specific P. humuli molecular markers such as Cox2 or ''Hex34. Cox2 was the first identified molecular probe that distinguishes P. humuli from P. cubensis'', a cucurbit Downy Mildew [3]. The probe uses SNP, single nucleotide polymorphism, on the Cox2 gene which has a unique genetic sequence for both P. humuli and P. cubensis. This allows efficient amplification of only one species of Downy Mildew, this case P. humuli, preventing misidentification with similar species. Once the PCR assay has been completed the samples are loaded into an Electrophoresis gel along with a 100 Kb DNA ladder and run for 60 minutes. When the run has been completed the samples can be photographed and measured against the ladder and positive controls of P. humuli that had been amplified along with the field samples. The results will provide a visual band of amplified DNA at the same position of the DNA ladder as the positive controls if the field samples contain P. humuli DNA.

Limitations

PCR assays don’t record the amplification of each sample through the entire degradation, elongation, and annealing process. Until the sample has been measured using a nano dropper or qBit machine there is now way to determine how much DNA is present. Even is a sufficient amount of DNA is extracted (20ng/ul >) there is no way to identify what kind of DNA has been amplified until a gel has been run. Also making and running a gel is time consuming and inconclusive results are a common occurrence. This delay prevents quick responses to possible Hop Infections on commercial farms as the species of Downy Mildew is needed to determine the correct fungicide application.

qPCR identification

This method of identification allows for quick identification of the target DNA by providing measurable results of the amplification process over the entire protocol. The target DNA is amplified using a set of 5’ probes that have an added fluorescent identification tag [3]. When one of the probes binds to the targeted sight on the P. humuli DNA it releases a unique light which is recorded by the machine. In the case of P. humuli the SNP probe Cox2 is not specific enough to be run without assisting annealing proteins. When performing qPCR, LNA proteins are added to the Cox2 probe which allows tight binding among the DNA bases. The added probes raise the melting temperature of the Cox2 complex which removes all incorrectly bonded probes by melting them during the denaturing step. Over the course of 90 minutes the protocol runs following a consistent pattern of a short denaturing step followed by a longer annealing step. A typical cycle runs for 35 cycles which providing sufficient amplification of the target DNA within the samples. The results are then present Cq values for each sample along with R2 and efficiency values. The Cq results indicate the amount of DNA that was amplified throughout the protocol.

Limitations

The assay has a difficult time amplifying samples gathered from the field because specific probes don’t work efficiently when there are numerous sources of DNA. The machine requires purified DNA that has been run through an extraction process like CTAB. But most samples extracted with CTAB still contain significant amounts of alternative DNA which causes cross reactivity, a process that prevents adequate amplification of P. humuli. This problem is most pronounced in soil and rhizome samples that However, the process still will run into obstacles that running field samples cross reactivity, the development of in mixed sample causes issues with definitive identification of HDM.

References

Bustin, S.A., Benes, V., Garson, J.A., Hellemans, J., Huggett, J., Kubista, M., Mueller, R., Nolan, T., Pfaffl, M.W., Shipley, G.L. and Vandesompele, J., 2009. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clinical chemistry, 55(4): 611-622.

Gent, D.H., Nelson, M.E., Farnsworth, J.L. and Grove, G.G., 2009. PCR detection of Pseudoperonospora humuli in air samples from hop yards. Plant pathology, 58(6): 1081-1091.

Summers, C. F., Adair, N. L., Gent, D. H., McGrath, M. T., & Smart, C. D. (2015). Pseudoperonospora cubensis and P. humuli detection using species-specific probes and high definition melt curve analysis. Canadian Journal of Plant Pathology, 37(3), 315-330.