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PCR Phytodiagnosis Kit. Diagnosis of turf diseases by microscopy and polymerase chain reaction (PCR).

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Javier M├ęndez Lorente
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Table of contents: PCR Phytodiagnosis Kit. Diagnosis of turf diseases by microscopy and polymerase chain reaction (PCR).

This article provides an insight into the methodology of turf disease diagnosis, analysing the efficacy of the PCR Phytodiagnosis Kit of Tiloom. The study analysed infected perennial ryegrass from a sports field at the Leicester Football Club by microscopy and a molecular technique. The process started with visual inspections in the field and microscopy, the analysis was concluded by quantitative real-time PCR. As a result, 11 common cold season pathogens were ruled out, with four positive identifications at various levels of infection, from weak positive to strong positive. The findings of these methods are discussed below.

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Bottom of the sports turf surface. 

The 100 % perennial ryegrass turf under investigation was mowed several times a week at 25 mm (actual). The previous week about 50 kg N/ha, 15 kg P/ha, 45 kg K/ha with an amino acid and algae were applied. This field was subjected to about 6 hours of football training per week.

Microscope analysis

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A site for infection. Mower injury on perennial ryegrass from the underside of the leaf.

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Infection and dieback of cut-leaf evergreen ryegrass under the dissecting microscope. Lesions caused by mowing appear to be a site of dieback and infection. Blackening and yellowing indicated signs of infected leaf material.

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Yellowing and twisted leaves were observed on several of the evergreen ryegrass leaves inspected. More evident was leaf dieback from the tip with whitish-grey structures on dying, wilted and twisted leaves of perennial ryegrass.

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Under an optical microscope, the filamentous structures were observed more closely.

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A filamentous bacterial infection stained 40x for visibility was observed within the leaves of perennial ryegrass.

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Bacterial filaments stained at 25x with aniline blue for display within the evergreen ryegrass leaf. The filamentous bacteria did not branch out and run longitudinally along the length of the sheet. 

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Filamentous bacteria at 40x stained with aniline blue within a perennial ryegrass leaf. Aggregated bacteria are not branched and have no apparent partitions (dividing sections/walls).

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On a different plant within the sampled area, under a dissecting microscope, cottony mycelium is visible protruding from the dying leaf material of perennial ryegrass.

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Branched hyphae, stained blue under light microscopy 25x

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Blue-tinged branching hyphae within the perennial ryegrass; unfortunately, a slightly unfocused T-shaped branching was observed.

Microscopy results

Light microscopy work identified a potentially common filamentous bacterial infection, for example, possibly early signs of a cyanobacteria (algal infection). On a different plant from the same sample, the protruding mycelium was observed more closely with observable branching in the form of a T. Characteristic of Rhizoctonia spp. Only with further molecular analysis could this be elucidated. 

DNA analysis

The polymerase chain reaction (PCR) is a molecular technique that can be used to detect the presence of target DNA. It is very accurate and detection can be identified in very small quantities. We now use this technique to identify the presence of plant/grass pathogens. These pathogens can be fungal or non-fungal (e.g. bacterial). The target DNA template (known as a primer or buffer) is required for DNA amplification. This method uses multiple DNA primers in PCR. This is known as multiplex PCR.

The DNA pathogens tested included: Bipolaris spp, Clarireedia spp, Colletotrichum spp., Drechslera spp., Fusarium poae , Gaeumannomyces spp, Laetisaria fuciformis , Microdochium nivale , Pyricularia grisea , Pythium spp, Rhizoctonia cerealis , R. solani , Sclerophthora macrospora. , Xanthomonas translucens , Labyrinthula spp. 

Or commonly known as: Leaf spot (Bipolaris & Dreschlera spp), dollar spot, anthracnose, Fusarium, take-all patch, red thread, Microdochium patch, grey leaf spot, Pythium (damping off), yellow patch, brown patch, yellow tuft (downy mildew), bacterial wilt, Rapid blight(rapid smut). 

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Six perennial ryegrass plants were sampled and prepared for further forensic analysis by polymerase chain reaction (PCR).

Perennial ryegrass leaf, shoot and root material was cut and homogenised for analysis.

The initial step of DNA extraction was the decomposition of cells from leaf, shoot and root material.

The extracted plant and the pathogen DNA concentrate.

The extracted DNA was washed and eluted in preparation for DNA amplification by PCR.

Microtubes containing PCR reaction solution for the 15 pathogens with amplification control.

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Four pathogens were positively detected by real-time quantitative PCR: Microdochium nivale (Fusarium patch), Pythium spp. (damping off), Rhizoctonia solani (brown patch) and R. cerealis (Yellow Patch). A high level of R. cerealis was detected.

PCR results

As the DNA analysed here did not include Cyanobacteria spp. it was not possible to confirm their presence. However, this multiplex PCR technique ruled out 11 other pathogens from the sampled turf. Although there were no obvious disease symptoms that could be associated with Microdochium/Fusarium Patch ( Microdochium nivale ) o Pythium hyphae characteristic of Yellow Patch ( Rhizoctonia cerealis ) or Brown Patch ( R. solani ) at the time of sampling. DNA analysis provided evidence of its presence. Interestingly, Yellow Patch disease was suspected to be the cause of the patches in the previous summer and autumn (July-October) of 2021 and 2023. It is known that rhizoctonia overwinters within the grass or plant debris. As the sampled ryegrass included leaves, roots, shoots and decomposed leaf material, it is likely that overwintering fungi were included in the reaction.

Typical symptoms of yellow spot infection occurring in the summer months of 2022 and 2023 (this photo is from August 2023) at the site.

Recommendations

It is important to note that multiple pathogens were identified in these samples. Normally, we name and discuss a disease when we observe signs and symptoms in turf. In this case at least four or five pathogens are involved. The pathogens identified may have caused yellowing and dieback with secondary bacterial infection. This is possibly typical, and the interaction between these pathogens and other disorders such as algae is having a combined effect on the condition of the turf; this probably requires further investigation.

Recommendations to control these threats to turfgrass health and condition would include a review of biological and physical approaches. For example, it is known that certain species of bacteria Bacillus control Rhizoctonia . There are a range of products available in the turf market. Early intervention at this time may have greater efficacy, as is usual with biological controls. For infection by cyanobacteria a sequential application of UV-C light is carried out, on the premise that algae are sensitive to UV rays. The application and dosage of nitrogen and phosphorus may also be reviewed to ensure sufficient levels. 

Diagnosing turf diseases is an important step in efficient and effective turf management. Whether it is allocating resources and budget, or acting efficiently and effectively to protect the environment while maintaining quality playing surfaces. We know that preventative disease management is often cost effective and provides good quality playing surfaces. 

While microscopy allowed a broad investigation of the infection, it could not identify the exact pathogen. Microscopic inspection was an opportunity to investigate the sites of infection and observe any characteristics of the pathogenic fungus, as shown by the mower-damaged cut leaves and the onset of infection. This was an important initial step towards investigating probable cause, should precise identification be required. 

The PCR technique was accurate in detecting pathogens present, whether these pathogens were symptomatically or latently infecting the turf. It may have provided an almost predictive indicator of possible future disease. This means that an integrated treatment plan for these specific pathogens can be initiated and continuously monitored. 

Accurate identification of pathogens in the symptomatic or dormant phase is best diagnosed using microscopy and molecular analysis, as shown in this case study. These techniques are likely to provide the greenkeeper with the best information for immediate action and disease management planning. In essence, these appear to be the best first steps for proactive and preventive integrated disease management. 

Contact Tiloom at info@tiloom.com and in less than 48 hours you will have the answer through a qPCR diagnostic test.

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