Mar 14, 2024
Lecanosticta acicola isolation protocol
- Colton Meinecke1,
- Caterina Villari1
- 1University of Georgia
Document Citation: Colton Meinecke, Caterina Villari 2024. Lecanosticta acicola isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g7pqe1gwz/v1
License: This is an open access document distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Created: March 14, 2024
Last Modified: March 14, 2024
Document Integer ID: 96679
Abstract
A guide to the reproduceable isolation of Lecanosticta acicola, the causal agent of brown spot needle blight of pines.
Lecanosticta acicola is a well-known aggressive foliar pathogen that attacks pines, causing brown spot
needle blight. Although it is a native pathogen of Pinus palustris in the southeast United States and an important pathogen of Pinus sylvestris Christmas tree farms, its behavior on many other Pinus species is poorly understood. Lecanosticta acicola is increasingly reported in associated with outbreak-scale damage on native pines, including Pinus taeda and Pinus strobus, throughout southern and eastern North America. The fungus is notoriously difficult to isolate in culture due to its slow growth and poor ability to compete with saprophytic microorganisms on nutrient-rich culture media. This protocol describes a reproducible method to obtain L. acicola isolates from Pinus taeda foliage for further study.
Needles were obtained from trees that displayed visible BSNB symptoms, including necrotic spots and bands bordered by yellow chlorotic zones and dead needle tips (Figure 1). Needles were incubated in a moist chamber (Ziploc bag with a damp paper towel) for at least 24 hours. Samples that were not overgrown with saprophytic fungi were allowed to incubate for several weeks. Chlorotic and necrotic spots and bands of individual needles were inspected for evidence of fungal fruiting, black acervuli erupting from the surface of the needle (EPPO Standards, 2015).
Figure 1. Pinus taeda foliage displaying brown spot needle blight symptoms,
including the namesake necrotic spots.
Fruiting bodies were excised from the needle with a sterile scalpel and transferred to a glass microscope slide. Slides were prepared by wiping with 70% EtOH, drying, and then placing one drop of sterile DI water in the center. The fruiting body was placed in the sterile water and crushed gently with a coverslip. The slide was then examined under a compound microscope (Figure 2). Acervuli and conidia morphologies were compared to references (EPPO Standards, 2015; CABI; van der Nest et al., 2019). In most cases, acervuli were observed emerging from within or just outside old bands and always from necrotic tissue. Microscope slides that contained fruiting structures similar to L. acicola were retained for isolation.
Figure 2. Lecanosticta acicola conidia emerging from an acervulus that formed within Pinus taeda foliage, viewed at 400 x magnification.
The coverslip was then carefully removed, leaving the water on the glass slide. A sterile inoculating loop was used to capture the fruiting body and a small amount of water. This suspension was then streaked for isolation (as with yeast or bacteria) on 2% malt extract agar (MEA) (Modified from Mullett and Barnes, 2012). A second loopful was streaked on ground pine needle agar (gPNA, modified from Luchi et al., 2007) prepared with 2% w/v agar and 8% w/v ground Pinus palustris needles. Plates were wrapped in parafilm and incubated on an open shelf under ambient conditions. Plates were inspected for growth once daily. Some single spore Lecanosticta colonies became visible after two days, and all were apparent within seven days after inoculation. Nascent colonies displayed hyaline or white aerial hyphae that turned gray or olive in approximately nine days on gPNA (Figure 3). Pigmentation occurred later in colonies on MEA, within 12 days, and was generally lighter (Figure 4). Notably, some colonies on gPNA appear to release a compound that turned the medium around the colonies an orange-red (Figure 3). Similar results were observed on gPNA plates prepared from P. elliotti and P. strobus needles. Lecanosticta acicola colonies grew radially at a rate of 2.5-3 mm per week, as previously reported (CABI). Very little contamination was observed, and what appeared was easily distinguished from Lecanosticta by its faster growth or yeast-like colony morphology.
Figure 3. Lecanosticta acicola colonies on gPNA at 5 days (A) and 8 days (B), highlighting colony pigmentation and discoloration of the surrounding media, and colonies on MEA at 4 days (C) and 12 days (D).
Protocol references
CABI. Retrieved 2022. Mycosphaerella dearnessii (brown spot needle blight). Invasive Species Compendium Datasheet 49057. Accessed online on 4/5/2022 (www.cabi.org/isc/datasheet/49057).
EPPO Standards. 2015. PM 7/46 (3) Lecanosticta acicola (formerly Mycosphaerella dearnessii), Dothistroma septosporum (formerly Mycosphaerella pini) and Dothistroma pini. EPPO Bulletin 45(2):163-182. DOI:10.1111/epp.12217
Luchi N., Capretti P., Bonello P. 2007. Production of Diplodia scrobiculata and Diplodia pinea pycnidia on ground Austrian pine needle agar medium. Phytopathologia Mediterranea 46(2):230-235.
Mullett M., Barnes I. 2012. Dothistroma: isolation and molecular identification methods. Determining Invasiveness and Risk of Dothistroma Training School May 2012, Brno, Czech Republic Detection and Diagnostics of Dothistroma. COST ACTION FP1102.
van der Nest A., Wingfield M.J., Janoušek J., Barnes I. 2019. Lecanosticta acicola: a growing threat to expanding global pine forests and plantations. Molecular Plant Pathology 20(10):1327-1364.