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Angular leaf spot (ALS) is caused by the bacterium Xanthomonas fragariae and is a common disease in strawberry. It was first described in 1962 by Kennedy and King, and strawberry is the only known host. The pathogen is transmitted from to fruit-production fields mainly through latent infections on asymptomatic nursery stock. Consequently, the pathogen has its greatest economic impact on the nursery industry, where the export of transplants is heavily regulated in many countries. Once in a planting, secondary spread occur from water dispersion caused by overhead irrigation and rainfall or mechanically from harvesting and maintenance procedures.
Symptoms
The foliage and the calyx of the plant is where symptoms commonly occur. The symptoms appear as small water-soaked lesions that typically begin on the bottom of the leaf (A). Lesions will enlarge over time to form angular spots that are restricted by the surrounding small veins. The lesions are translucent when viewed with transmitted light (B) but dark green when viewed with reflected light (C). As the lesions age, they will eventually be visible from the top of the leaf as irregular reddish-brown spots with a yellow halo (D). When the infection has reached this point, it can be sometimes difficult to distinguish ALS symptoms from those caused by common leaf spot (caused by Mycosphaerella fragariae) and leaf scorch (caused by Diplocarpon earliana). The bacterium can also infect the calyx (E). When infection is severe, the calyx will die causing “black cap” which significantly reduces the marketability of the fruit. Under moist conditions, usually in the morning when the dew is still present, the lesions can form a viscous bacterial exudate on the lower leaf surface that will dry into a scaly whitish film (F). The bacterium can also travel systemically through the vascular system and infect the crown, other leaf tissue, and daughter plants. This was once thought to play a significant role in the infection process, but has since been questioned based on studies tracking the bacteria in the plant following several different modes of infection (Wang and Turechek, 2018).
Morphology
Xanthomonas fragariae is an aerobic, slow growing, gram-negative, non-capsulate rod bacterium, 0.4 x 1.3 mm with a single polar flagellum. X. fragariae is a slow-growing bacterium and when grown on sucrose peptone agar (SPA), the colonies appear initially as white, glistening pinpoint colonies and ultimately form yellow mucoid colonies after 5-7 days.
Disease Cycle
The primary inoculum of Xanthomonas fragariae usually comes from infected tissues in nursery transplants or dried plant tissue in the field. X. fragariae is resistant to desiccation and can live through various harsh weather conditions in both the summer (typical of Florida production) and winter (elsewhere in the US). It is also known to survive on other non-plant surfaces like plastic and cardboard for extended periods of time. Secondary spread of X. fragariae occurs from overhead irrigation and rainfall. The motile cells are contained in the water droplets. The bacteria can also be passed manually by blossom removal (nurseries), harvesting, and maintenance operations. Young leaf tissue and leaves, on healthy plants are more likely to become infected over older leaves or environmentally stressed plants. Infection is favored by moderate temperatures – between 10°C and 30°C – and when surface moisture is present. Leaves do not need to be wet for extended periods for infection to occur; however, long wetting periods will result in more infection assuming inoculum is present.
Control
Antibiotics and copper-containing pesticides, such as streptomycin sulfate, oxytetracycline, cupric hydroxide, and copper ammonium carbonate, have been shown to be effective protectants against ALS, however, in some instances copper products may be phytotoxic. Heat treatment of nursery plants prior to planting has been shown to reduce and/or delay epidemic development of ALS in the field. Only F. moschata has been shown to be immune to ALS. Wild octoploid species and cultivars of F. × ananassa vary greatly in susceptibility to X. fragariae, indicating that, in this gene pool, it may be possible to select clones with increasing degrees of resistance. However, no important commercial cultivars have been shown to be immune to ALS under field conditions.
Culture and Molecular Detection Methods
Currently, field inspections for symptoms are used to certify plants free of ALS but visual inspection is not useful for detecting plants infected systemically. There are several methods of detection of Xanthomonas fragariae.
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X. fragariae can be grown on several media including Sucrose Peptone Agar (SPA), Yeast Dextrose Carbonate Agar (YDC), Yeast Peptone Glucose Agar (YPGA), and Wilbrinks-N media. We routinely use SPA for growing X. fragariae. The bacteria are grown at 25oC for 5-7 days. The colonies will appear initially as white, glistening pinpoint colonies and ultimately form yellow mucoid colonies. X. fragariae grown on YDC media is grown at 20°C for 5-7 days. Colonies will appear as a yellow color, glistening smooth surface with sticky colonies. Bacteria grown on YPGA and Wilbrinks-N media is grown at 25oC for 5-7 days. Colonies will appear yellow in color, circular, slightly convex, smooth and mucoid.
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Conventional, nested, and real-time PCR primers have been developed for rapid detection and identification of Xanthomonas fragariae. For standard PCR applications, the most common sets of primers were developed by Hartung and Pooler (1997) and are named 241, 245, and 295. Primers for nested PCR were developed by Zimmerman et al. (2004). They used a fragment from the 245 primers and made an internal set of primers called 245.5 and 245.267. A real-time Taqman assay was developed by Turechek, Hartung, and McCallister (2008). The real-time assay has become the preferred method of detection due to its speed and accuracy. The primers (q241, q245, and q295) each share one of the conventional PCR primers (241, 245, and 295) and each primer set was paired with a probe. The information on sensitivity and specificity of the primer sets was used to evaluate the performance of these primers with ROC cure analysis under different tolerances for the disease. The results of this analysis can be used to provide guidance on threshold selection to manage disease below unacceptable levels.
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ELISA (Enzyme-Linked Immunosorbent Assay) kits are available for X. fragariae from Bioreba (Reinach, Switzerland) and Loewe (Sauerlach, Germany). ELISA uses cell suspensions or homogenized plant material for the test. Unlike the other methods, ELISA detection uses antibodies to detect proteins in the sample not DNA. This method of detection typically takes 2 days to complete.
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A LAMP (Loop-Mediated Isothermal Amplification) assay for sensitive and specific detection of X. fragariae was developed by Wang and Turechek (2016). LAMP amplifies DNA at a single, constant temperature, thus removing the need for expensive thermal cyclers. You can view the LAMP samples by gel electrophoresis, fluorescent dyes (SYTO 9, Thermo Fisher), or by adding HNB (hydroxynaphthol blue) to the reaction. Using HNB will give you a colorimetric assay where the positive samples are blue and the negatives are purple. LAMP is particularly suited for field-based pathogen detection.
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RPA (Recombinase Polymerase Amplification) technology is another method that uses a single temperature to run the reaction, making it also useful for field use. RPA kits are made by Twist DX (Maidenhead, United Kingdom). In the RPA reaction, enzymes are used to bind the primers to the DNA which amplifies the corresponding fragment only if it is present. Our assay utilizes the existing real-time primers and the final reaction can be viewed on an agarose gel or if SYBR Green I (Thermo Fisher) is added it can be viewed under a UV light.
Presentations and Posters
A quantitative PCR assay for detection of Xanthomonas fragariae
Hot water treatment as a means to eradicating X. fragariae from strawberry nursery stock
A Concise Introduction to Receiver Operating Characteristic (ROC) Curve Analysis
North American Strawberry Growers Association 2019 Presentation
Selected Sources
Hildebrand, D. C., Schroth, M. N., and Wilhelm, S. 1967. Systemic invasion of strawberry by Xanthomonas fragariae causing vascular collapse. Phytopathology 57:1260–1261.
Kennedy, B. W., and King, T. H. 1962. Angular leaf spot of strawberry caused by Xanthomonas fragariae sp. nov. Phytopathology 52:873–875.
Maas, J. L., Pooler, M. R., and Galletta, G. J. 1995. Bacterial angular leafspot disease of strawberry: Present status and prospects for control. Adv. Strawberry Res. 14:18–24.
Smith, I. M., McNamar, D. G., Scott, P. R., and Harris, K. M., eds. 1992. Xanthomonas fragariae. Pages 829-833 in: Quarantine Pests for Europe. Data Sheets on European Communities and for the European and Mediterranean Plant Protection Organization. CAB International, Wallingford, UK.
Thind, B S. Phytopathogenic Bacteria and Plant Diseases. Boca Raton, FL. Taylor and Francis Group. 2020.
