Case Study: turfgrass disease diagnosis by microscopy & polymerase chain-reaction (PCR)

Introduction

This article is an insight into turfgrass disease diagnosis methodology at the Sports Turf Academy. Here, infected perennial ryegrass from a sports pitch was analysed by microscopy and a molecular technique. The process started with visual inspections in the field and microscopy, the analysis was concluded following real-time quantitative PCR. The output of which, ruled out 11 common cool-season pathogens, with four positive identifications at various levels of infection from weak to strong positive. The findings of these methods are discussed below.

Background of the sports turf surface

The 100 per cent perennial ryegrass pitch under investigation was cut multiple times a week at 25 mm (actual). In the previous week, about 50 kg N / ha, 15 Kg P / ha, 45 kg K / ha with an amino-acid and seaweed was applied. This pitch was subjected to about 6 hours of football training per week.

Microscope Analysis

Microscopy Results

The light microscopy work identified a potentially common filamentous bacterial infection e.g. possibly early signs of a Cyanobacteria (algal-infection). In a different plant from the same sample, protruding mycelium was observed more closely with observable T-like branching. Characteristic of Rhizoctonia spp. Only with further molecular analysis could this be elucidated.

DNA Analysis

Polymerase chain-reaction (PCR) is molecular technique that may be used to screen for the presence of target DNA. It is highly accurate and detection can be identified in the smallest of quantities. We now utilise this technique for identifying the presence of plant / turfgrass pathogens. These pathogens may be fungal, or non-fungal (e.g. bacterial). The template of the targeted DNA (known as a primer) is needed for DNA amplification. This method uses multiple DNA primers in the PCR. This is known as multiplex PCR.

DNA pathogens screened 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.

PCR Results

As DNA screened here did not include Cyanobacteria spp. it was not possible to confirm its presence. This multiplex PCR technique, however, 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) or Pythium, hyphae characteristic of Yellow Patch (Rhizoctonia cerealis) or Brown Patch (R. solani) at time of sampling was observed by microscopy. The DNA analysis provided evidence of their presence. Interestingly, Yellow Patch disease was suspected as the cause of patches in the previous summer and autumn (July-October) in 2021 and 2023. Rhizoctonia is known to over-winter within the turf/plant debris. As the sampled ryegrass included leaf, root, shoot, leaf decayed material it is likely over-wintering fungi was included in the reaction.

Findings

It's important to note that multiple pathogens were identified within these samples. Typically, we often name and discuss one disease when we observe signs and symptoms in the turf. In this case, at least four or five pathogenic agents are involved. The identified pathogens may have caused the yellowing and die-back with bacterial secondary-infection. Possibly, this is typical, and the interaction between these pathogens and other disorders such as algae are having a combined affect on turfgrass condition, this likely requires further research.

The recommendations for controlling these threats to turfgrass health and condition would include reviewing biological and physical approaches. For example, certain species of Bacillus bacteria are known to control Rhizoctonia. There are a range of products available on the turf market. Early intervention at this time may have a greater efficacy, as is typical with biological controls. For the Cyanobacteria infection a sequential application of UV-C light, under the premise that algae are UV-sensitive. The application and rate of nitrogen and phosphorus may also be reviewed, with the view to ensure sufficient levels.

Diagnosing turfgrass diseases is an important step in efficient and effective turfgrass management. Whether it is allocation of resources and budget, or acting efficiently and effectively to protect the environment while maintain quality playing surfaces. We know preventative disease management is often cost-effective and facilitates good quality playing surfaces.

While the microscopy allowed a wide-ranging investigation of the infection, it could not pin-point the exact pathogenic agent. The microscope inspection was an opportunity to investigate infection sites and note any characteristics of the fungal-pathogen, as shown with the damaged mower cut leaves and the infection on-set. This was an important initial step toward investigating a likely cause --should the extent of precise identification be required.

The PCR technique was accurate in detecting pathogens that were present, whether these pathogens were symptomatically infecting the turf or latent. It may have provided an almost predictive indicator for future likely diseases. This means an integrated plan of treatments for these specific pathogens may begin and be continuously monitored.

The accurate identification of pathogens in the symptomatic or dormant phase may be best diagnosed by utilising both microscopy and molecular analysis, as given in this case study. These techniques would likely give the turf manager the best information for immediate action and planning ahead with disease management. In essence, these seem to be the best first steps of proactive and preventative integrated disease management.

Acknowledged thanks to Jamie Reading at LCFC for initiating this investigation and to Kate Entwistle (Turfgrass Disease Center) for assistance with the Cyanobacteria identification.

Please feel free to DM me or email [email protected] if you would like to know more about this type of turfgrass disease analysis work.