A Field-Portable Diagnostic Approach Confirms Laurel Wilt Disease Diagnosis in Minutes Instead of Days

2021 ◽  
Vol 47 (3) ◽  
pp. 98-109
Author(s):  
Jeffrey Hamilton ◽  
Stephen Fraedrich ◽  
Campbell Nairn ◽  
Albert Mayfield ◽  
Caterina Villari
Keyword(s):  
Lamp Assay ◽  
Disease Diagnosis ◽  
Wilt Disease ◽  
Diagnostic Process ◽  
Laurel Wilt ◽  
Xyleborus Glabratus ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease ◽  
Persea Borbonia ◽  
Species Specific

Background: Laurel wilt disease has caused the extensive mortality of lauraceous species in the southeastern United States. The causal agent is an invasive fungus, Raffaelea lauricola, which is a symbiont of the beetle Xyleborus glabratus and causes a rapid, fatal vascular wilt. Early diagnosis of laurel wilt is imperative for efficient disease management. The current diagnostic process, however, is slow due to the lengthy laboratory procedures required to confirm pathogen presence. Methods: We tested the robustness and field-portability of a recently developed, species-specific, loop-mediated isothermal amplification (LAMP) assay for R. lauricola, with the overall goal of eliminating the need for a laboratory confirmation of the diagnosis. We tested the robustness of the assay using benchtop equipment with naturally infected samples. We then tested the assay directly in the field using a portable device. Results: The assay successfully detected R. lauricola directly from symptomatic wood tissue using crude DNA extracts. Furthermore, the assay readily allowed users to distinguish between symptoms caused by R. lauricola infection and similar symptoms caused by other agents. In-field, we assayed wood samples from symptomatic redbay (Persea borbonia [L.] Spreng) and sassafras (Sassafras albidum [Nutt.] Nees) across the Southeast and successfully detected R. lauricola-infected trees in less than an hour. Conclusion: Results of this study confirmed that the field-deployable LAMP assay is robust and can rapidly and accurately detect R. lauricola in infected trees directly on-site. LAMP technology is well suited for in-field implementation, and these results serve as an incentive for further development and use of this technology in the field of forest pathology.

scholarly journals Potential Economic Impact of Laurel Wilt Disease on the Florida Avocado Industry

2010 ◽  
Vol 20 (1) ◽  
pp. 234-238 ◽  
Author(s):  
Edward A. Evans ◽  
Jonathan Crane ◽  
Alan Hodges ◽  
Jason L. Osborne
Keyword(s):  
Regional Economy ◽  
Impact Analysis ◽  
Control Measure ◽  
Wilt Disease ◽  
Persea Americana ◽  
Economic Losses ◽  
Laurel Wilt ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease ◽  
The Impact

This article describes and provides preliminary estimates of the potential economic losses that could result from an incursion of the recently discovered exotic laurel wilt disease caused by Raffaelea lauricola, in the main avocado (Persea americana) growing area of Florida. Estimates are provided for the direct losses as well as the indirect or “spillover” losses that could occur across the rest of the regional economy. The Impact Analysis for Planning (IMPLAN) input-output multipliers were used in assessing the regional impacts. The results of the investigation indicate that the direct loss to the industry in terms of lost sales, property damage, and increased management costs could range from $356 million in a do-nothing situation to about $183 million if damage control measure were 50% effective. If increased management costs and decreased property values are ignored, the adverse impact on the regional economy could range from $54 million in a do-nothing situation to $27 million in a case in which the treatments result in only a 50% reduction in avocado production.

scholarly journals First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Sassafras in North Carolina

Plant Disease ◽  
2019 ◽  
Vol 103 (1) ◽  
pp. 155-155 ◽  
Author(s):  
A. E. Mayfield ◽  
C. Villari ◽  
J. L. Hamilton ◽  
J. Slye ◽  
W. Langston ◽  
...  
Keyword(s):  
North Carolina ◽  
Wilt Disease ◽  
Laurel Wilt ◽  
First Report ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease

First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Swamp Bay in Louisiana

Plant Disease ◽  
2019 ◽  
Vol 103 (7) ◽  
pp. 1777
Author(s):  
R. Olatinwo ◽  
J. Hwang ◽  
W. Johnson ◽  
S. W. Fraedrich
Keyword(s):  
Wilt Disease ◽  
Laurel Wilt ◽  
First Report ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease

scholarly journals First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Spicebush in Louisiana

Plant Disease ◽  
2021 ◽  
Author(s):  
Rabiu Olatinwo ◽  
Jaesoon Hwang ◽  
Wood Johnson
Keyword(s):  
United States ◽  
Large Subunit ◽  
Morphological Characteristics ◽  
Wilt Disease ◽  
Persea Americana ◽  
Food Sources ◽  
Laurel Wilt ◽  
First Report ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease

In the past two decades, laurel wilt disease has significantly affected members of the Lauraceae in the southeast United States, causing widespread mortality of native redbay (Persea borbonia (L.) Spreng), and incidence of infections in avocado (Persea americana Mill.), sassafras (Sassafras albidum L.) and swamp bay (Persea palustris [Raf.] Sarg.) (Fraedrich et al., 2008, 2015, Olatinwo, et al. 2019). Laurel wilt is a vascular disease caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva), a fungus vectored by a non-native ambrosia beetle Xyleborus glabratus Eichhoff (Fraedrich et al. 2008). In August 2020, we investigated the mortality of a spicebush shrub (Lindera benzoin L.) (3.8 cm diameter at root collar, two m height) located ca. 17 mi northeast of Colfax, Grant Parish, Louisiana (31.750263° N, -92.643694° W). Evaluation of the dead shrub revealed brown, persistent foliage, and black vascular discoloration of the sapwood, typical symptoms of laurel wilt (Fig. S1). Although, beetle holes were observed on the sapwood, no beetle was found in galleries at the time. In the laboratory, a fungus consistently isolated from surface-sterilized sapwood tissues plated on potato dextrose agar (PDA) was identified as R. lauricola based on the morphological characteristics of the isolate (i.e., mucoid growth, conidiophores, and oblong/ovoid shape conidia [Harrington et al. 2008]). The fungal isolate was denoted as SB1. The identity of the fungus was confirmed by positive PCR amplification of the large subunit ribosomal RNA gene region using species-specific primers; rab-lsu-rl_F: CCCTCGCGGCGTATTATAG and rab-lsu-rl_R: GCGGGGCTCCTACTCAAA (Olatinwo, unpublished). The sequence of the isolate SB1 (GenBank Accession no. MW207371) showed 100% homology to the R. lauricola strain CBS 127349 sequence (GenBank Accession no. MH877933). The pathogenicity of SB1 on spicebush was evaluated on four healthy shrubs (average: 1 m height and 40 mm in diameter) at the location from which the original detection was made. Stems of two spicebush shrubs were inoculated with SB1 agar plugs from a 14-day old culture on PDA, while plain PDA plugs were used on the remaining two shrubs as non-inoculated controls. Agar plugs were placed in 5 mm (0.2 in) diameter hole punched on the bark with cork-borer as described by Mayfield et al (2008). After six weeks, the R. lauricola inoculated shrubs were wilted with noticeable blackened tissue discoloration in the sapwood, while the control trees remained healthy (Fig. S2). Raffaelea lauricola was re-isolated from tissue of the two inoculated, symptomatic shrubs, but not from the control trees. The sequence of the re-isolated R. lauricola isolate, denoted as SB3 (GenBank Accession no. MW207372), showed 100% homology to the R. lauricola strain CBS 127349 and isolate SB1. This first documentation of laurel wilt on spicebush in Louisiana is significant because, spicebush berries, leaves, and twigs are food sources for forest animals, birds, and insects including whitetail deer and spicebush swallowtail (Papilio troilus L.). Since its first report on sassafras in 2014 (Fraedrich et al. 2015), laurel wilt has spread across Louisiana on sassafras and swamp bay (Olatinwo et al. 2019) and has been confirmed in14 parishes. This report shows the relentless nature of the disease, as the pathogen moves from one vulnerable host to the next, expanding into new locations and threatening forest ecosystems across the southern United States.

scholarly journals First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Pondspice in Florida

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1588-1588 ◽  
Author(s):  
M. Hughes ◽  
J. A. Smith ◽  
A. E. Mayfield ◽  
M. C. Minno ◽  
K. Shin
Keyword(s):  
Small Subunit ◽  
Wilt Disease ◽  
Spore Suspension ◽  
Ambrosia Beetle ◽  
Laurel Wilt ◽  
Mature Adult ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease ◽  
Redbay Ambrosia Beetle ◽  
Small Subunit Rdna

Laurel wilt is a fungal vascular disease of redbay (Persea borbonia (L.) Spreng) and other plants in the family Lauraceae in the southeastern United States (1). The disease is caused by Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, which is vectored by the exotic redbay ambrosia beetle (Xyleborus glabratus Eichhoff) (2). Pondspice (Litsea aestivalis (L.) Fern.) is an obligate wetland shrub listed as endangered in Florida and Maryland and threatened in Georgia (4). On 29 August 2008, 369 of 430 (85%) pondspice trees observed at St. Marks Pond in St. John's County, Florida were dead and/or dying (4). Stem samples were collected from plants with wilted and reddened foliage, entrance holes with boring dust characteristic of ambrosia beetle attack, and dark discoloration in the outer sapwood. Discolored stem sections were surface disinfested for 30 s in a 5% sodium hypochlorite solution and then plated onto cycloheximide streptomycin malt extract agar (1). Smooth, cream-buff, submerge hyphae with uneven margins resembling R. lauricola (2) was observed growing from all sapwood pieces. DNA was extracted from a single isolate (PL 392) and the 18s small subunit rDNA was PCR amplified and sequenced with primers NS1 and NS4 (3), resulting in a 1,026-bp amplicon. A BLASTn search showed identical homology to R. lauricola strain PL 159 (GenBank Accession No. EU257806). The 18s small subunit rDNA sequence was deposited into GenBank (FJ514097). In May 2011, a spore suspension was made by flooding a single-spore culture plate of isolate PL 392 with 2 ml of sterile water, collecting the spores by pipette, and quantification by hemacyometer to 1.5 × 106 spores/ml. Pathogenicity tests were conducted on 1 to 1.5 m tall pondspice plants. Six saplings were wounded by a 3/32-inch drill bit, with four receiving 50 μl of the spore suspension and two serving as water-inoculated controls. All plants were kept in a greenhouse under ambient temperature. Within 21 days, all fungal-inoculated saplings displayed complete canopy wilt, typical of laurel wilt. R. lauricola was later recovered from all four infected plants, completing Koch's postulates. To determine if the vector can reproduce in pondspice, infected stem sections were placed in a plastic rearing box indoors at room temperature, and both callow and mature adult female X. glabratus emerged in October and November 2008. Although laurel wilt has been previously observed on pondspice in South Carolina and Georgia (1), this is the first confirmation of the disease on pondspice in Florida and the first confirmation of the vector from stem material of this host. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) M. A. Innis et al. PCR Protocols, A Guide to Methods and Applications. Academic Press. San Diego, CA, 1990. (4) J. A. Surdick and A. M. Jenkins. Pondspice (Litsea aestivalis) Population Status and Response to Laurel Wilt Disease in Northeast Florida. Florida Natural Areas Inventory, Tallahassee, FL, 2009.

scholarly journals First Report of Laurel Wilt Disease Caused by Raffaelea lauricola on Silk Bay in Florida

Plant Disease ◽  
2012 ◽  
Vol 96 (6) ◽  
pp. 910-910 ◽  
Author(s):  
M. A. Hughes ◽  
K. Shin ◽  
J. Eickwort ◽  
J. A. Smith
Keyword(s):  
Fungal Growth ◽  
Wilt Disease ◽  
Spore Suspension ◽  
Sodium Hypochlorite Solution ◽  
Laurel Wilt ◽  
Fungal Isolation ◽  
Raffaelea Lauricola ◽  
Laurel Wilt Disease ◽  
Vascular Discoloration ◽  
Scrub Forests

Silk bay (Persea humilis Nash) is a member of the Lauraceae precinctive to the scrub forests of central and southern Florida and a sister species to the primary laurel wilt host, redbay (P. borbonia (L.) Spreng), which is generally not found in these ecosystems. In November 2011, observations of silk bay mortality near Lake Placid in Highlands County, FL, were reported to Florida Forest Service agents. A subsequent visit to roadside and homeowners' properties in the area revealed many dead and dying silkbays with characteristic laurel wilt symptoms, including wilted crowns with brown persistent foliage, frass accumulated at tree bases, sapwood with dark streaking, and ambrosia beetle entrance holes (1). Twig samples were taken and later confirmed as silk bay by the Florida Division of Plant Industry in Gainesville. Trunk samples were taken from four trees for fungal isolation. Stem sections with vascular discoloration were surface disinfested for 30 s in a 5% sodium hypochlorite solution and then plated onto cycloheximide streptomycin malt extract agar (CSMA) (1). All sapwood pieces from the four samples resulted in the same cream-buff submerged fungal growth characteristic of the laurel wilt pathogen Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva (2). DNA was extracted from a single-spore derived isolate, PL1389, and an 18S small subunit ribosomal RNA gene sequence was generated with primers NS1 and NS4, resulting in a 1,031-bp amplicon (3). A BLASTn search showed identical homology to R. lauricola strains PL159 and PL382 (GenBank Accessions No. EU257806 and JF797171, respectively, 100% similarity, e-value 0.0, and a total score of 1,982). The sequence was deposited into GenBank and assigned the accession No. JQ247569. In December 2011, a spore suspension was made by flooding a PL1389 culture plate with 2 ml of sterile water, collecting by pipette, and quantification and adjusting to 3.25 × 106 spores/ml by hemacytometer. Pathogenicity was tested on potted plants in a growth chamber experiment. Five silk bays and three redbays were drill-wounded with a 3/32” drill bit and inoculated with 20 μl of the spore suspension. Three silk bays and two redbays served as water-inoculated controls. Within 5 weeks, all inoculated plants displayed the wilt and vascular discoloration characteristic of laurel wilt disease, while all water-inoculated controls remained healthy. Sapwood samples from all plants were plated onto the same CSMA media. R. lauricola was later recovered only from the wilted plants inoculated with PL1389, while no fungal growth was recovered from the asymptomatic water-inoculated controls. Silk bay, which plays a significant role in the limited scrub ecosystems of Florida, has now become another host in the laurel wilt epidemic, with its implications upon the scrub forests yet to be seen. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) T. C. Harrington et al. Mycotaxon 104:399, 2008. (3) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press. San Diego, CA, 1990.

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