scholarly journals Development of Multilocus PCR Assays for Raffaelea lauricola, Causal Agent of Laurel Wilt Disease

Plant Disease ◽  
2014 ◽  
Vol 98 (3) ◽  
pp. 379-383 ◽  
Author(s):  
Tyler J. Dreaden ◽  
John M. Davis ◽  
Carrie L. Harmon ◽  
Randy C. Ploetz ◽  
Aaron J. Palmateer ◽  
...  

Laurel wilt, caused by the fungus Raffaelea lauricola, is an exotic disease that affects members of the Lauraceae plant family in the southeastern United States. The disease is spreading rapidly in native forests and is now found in commercial avocado groves in south Florida, where an accurate diagnostic method would improve disease management. A polymerase chain reaction (PCR) method based on amplifying the ribosomal small-subunit DNA, with a detection limit of 0.0001 ng, was found to be suitable for some quantitative PCR applications; however, it was not taxon specific. Genomic sequencing of R. lauricola was used to identify and develop primers to amplify two taxon-specific simple-sequence repeat (SSR) loci, which did not amplify from related taxa or host DNA. The new SSR loci PCR assay has a detection limit of 0.1 ng of R. lauricola DNA, is compatible with traditional and real-time PCR, was tested in four labs to confirm consistency, and reduces diagnostic time from 1 week to 1 day. Our work illustrates pitfalls to designing taxon-specific assays for new pathogens and that undescribed fungi can limit specificity.

Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1588-1588 ◽  
Author(s):  
M. Hughes ◽  
J. A. Smith ◽  
A. E. Mayfield ◽  
M. C. Minno ◽  
K. Shin

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.


Plant Disease ◽  
2014 ◽  
Vol 98 (4) ◽  
pp. 559-564 ◽  
Author(s):  
A. Jeyaprakash ◽  
D. A. Davison ◽  
T. S. Schubert

The laurel wilt disease fungus, Raffaelea lauricola, is killing redbay trees, spreading rapidly in the U.S. southeastern coastal plain forest, and posing a serious threat to the avocado industry in Florida. A molecular tool is urgently required to facilitate detection of this pathogen. The 5′ region of the large ribosomal RNA (28S) gene is highly variable among Raffaelea spp. and ideal for this purpose but amplification of this sequence from R. lauricola has been difficult. Different amplification conditions were tested and a high-fidelity polymerase chain reaction (PCR) procedure utilizing a dNTP mix containing 7-deaza-dGTP was found to reliably amplify 28S sequences from R. lauricola. Sequencing the amplified products or cloned inserts also turned out to be difficult and required using a custom-blended sequencing mix containing 1 M betaine, 5% dimethyl sulfoxide, and dGTP-BigDye v3.1. Three GC-rich stem and loop or cruciform secondary structures were discovered, which may have interfered with amplification. This improved protocol made it possible to partially characterize the internal transcribed spacers sequence from R. lauricola, which also has interfering secondary structures. A TaqMan real-time PCR assay was designed using the species-specific 28S sequences and this allowed detection of R. lauricola from wood tissues or cultures. Wood tissues from symptomatic redbay, avocado, and sassafras trees in Florida were screened using this TaqMan assay and several were found to test positive for R. lauricola. Results were further confirmed by performing Koch's postulates for avocado specimens collected from commercial grooves.


2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Mei Jean Sue ◽  
Swee Keong Yeap ◽  
Abdul Rahman Omar ◽  
Sheau Wei Tan

Polymerase chain reaction-enzyme linked immunosorbent assay (PCR-ELISA) is an immunodetection method that can quantify PCR product directly after immobilization of biotinylated DNA on a microplate. This method, which detects nucleic acid instead of protein, is a much more sensitive method compared to conventional PCR method, with shorter analytical time and lower detection limit. Its high specificity and sensitivity, together with its semiquantitative ability, give it a huge potential to serve as a powerful detection tool in various industries such as medical, veterinary, and agricultural industries. With the recent advances in PCR-ELISA, it is envisaged that the assay is more widely recognized for its fast and sensitive detection limit which could improve overall diagnostic time and quality.


Plant Disease ◽  
2009 ◽  
Vol 93 (10) ◽  
pp. 1079-1079 ◽  
Author(s):  
J. A. Smith ◽  
T. J. Dreaden ◽  
A. E. Mayfield ◽  
A. Boone ◽  
S. W. Fraedrich ◽  
...  

Laurel wilt disease, caused by Raffaelea lauricola (T.C. Harr., Fraedrich & Aghayeva sp. nov.), which is a fungal symbiont of the nonnative redbay ambrosia beetle (Xyleborus glabratus Eichhoff), has caused widespread mortality of native redbay (Persea borbonia (L.) Spreng) in Georgia, South Carolina, and Florida since 2002. The disease has been noted on other species in the Lauraceae including sassafras in Georgia (1), and more recently, on avocado and camphor in Florida (4). Since 2005, wilted shoots, branch dieback, and tree death have been observed in sassafras trees (Sassafras albidum (L.)) in Liberty, McIntosh, Chatham, Effingham, Bulloch, Evans, and Screven counties in Georgia; Bamberg, Beaufort, Charleston, Colleton, Hampton, and Orangeburg counties in South Carolina; and Putnam County in Florida. Symptomatic sassafras trees ranged from 1 to 12 m high and 2.5 to 25 cm in diameter at breast height. In contrast to red bay trees that retain wilted foliage, symptomatic sassafras defoliate rapidly as trees wilt and die. Multiple symptomatic ramets originating from a common root system have been observed. Removal of bark from stem and root sections from wilted trees revealed black-to-brownish staining in the sapwood, characteristic of laurel wilt. Wood chips from symptomatic areas of branches and roots were surface sterilized and plated on cycloheximide-streptomycin malt agar as previously described (1) and R. lauricola was routinely isolated. Small subunit (18S) sequences from rDNA were amplified by PCR and sequenced using primers NS1 and NS4 (3) for isolates from sassafras from Florida and South Carolina. BLASTn searches revealed homology to Raffaelea sp. C2203 (GenBank Accession No. EU123076, 100% similarity) described by Fraedrich et al. (1) from redbay and later named R. lauricola (2). The small subunit rDNA sequences for these isolates have been deposited into GenBank ( http://www.ncbi.nlm.nih.gov/Genbank/index.html ) and assigned Accession Nos. EU980448 (Florida) and GQ329704 (South Carolina). Koch's postulates have been completed with R. lauricola on this host previously (1). Laurel wilt on sassafras often was geographically isolated from other symptomatic hosts in Georgia and South Carolina and appears to occur on this host independently of proximity to redbay. Further studies to determine the epidemiology of laurel wilt on sassafras, potential resistance, and impact on sassafras life history and distribution are needed. Given the clonal nature of sassafras, the disease would appear to have the potential to move through roots of trees once established in a stand. 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. Smith et al. Plant Dis. 93:198, 2009.


2018 ◽  
Vol 28 (2) ◽  
pp. 102-108 ◽  
Author(s):  
Julian Mendel ◽  
Kenneth G. Furton ◽  
DeEtta Mills

Laurel wilt disease, incited by Raffaelea lauricola, has resulted in the death of more than 300 million laurel trees (Lauraceae) in the United States. One such tree is the commercially important avocado (Persea americana), the second largest tree crop in Florida other than citrus (Citrus sp.). This disease affects the industry in South Florida and two larger avocado industries in Mexico and California have taken notice. Trees succumb soon after infection, and once external symptoms are evident, the disease is very difficult to control and contain as the pathogen can spread to adjacent trees via root grafting. Presently, there is no viable, cost-effective method of early diagnosis and treatment. This study was undertaken to evaluate the use of scent-discriminating canines (Canis familiaris) for the detection of laurel wilt–affected wood from avocado trees. Three canines, one Belgian Malinois and two Dutch Shepherds, were trained and studied for this ability. In addition, prevailing weather conditions were recorded and evaluated to determine their effect on canine performance. The results of this evaluation indicated that canines can detect laurel wilt–affected wood and the laurel wilt pathogen and may be useful in the detection of laurel wilt–diseased trees in commercial groves.


2021 ◽  
Author(s):  
Stephen Fraedrich

Abstract Laurel wilt is responsible for the death of hundreds of millions of redbay (Persea borbonia sensu lato) trees throughout the southeastern USA, and the disease is also having significant effects on other species such as sassafras (Sassafras albidum) in natural ecosystems and avocado (Persea americana) in commercial production areas of south Florida. Laurel wilt is caused by the pathogen Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle, Xyleborus glabratus. Thus far, the disease is confined to members of the Lauraceae that are native to the USA, or native to such places as the Caribbean, Central America and Europe and grown in the USA. The beetle and fungus are native to Asia and were likely introduced with untreated solid wood packing material at Port Wentworth, Georgia in the early 2000s. Since that time laurel wilt has spread rapidly in the coastal plains of the southeastern USA, spreading north into central North Carolina, as far west as Texas, and reaching the southernmost counties of Florida. Current models suggest that X. glabratus can tolerate temperature conditions that occur throughout much of the eastern USA, and so the disease threatens sassafras throughout much of this region. The disease poses a threat to lauraceous species indigenous to other areas of the Americas as well as Europe and Africa.


2010 ◽  
Vol 20 (1) ◽  
pp. 234-238 ◽  
Author(s):  
Edward A. Evans ◽  
Jonathan Crane ◽  
Alan Hodges ◽  
Jason L. Osborne

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.


Plant Disease ◽  
2019 ◽  
Vol 103 (1) ◽  
pp. 155-155 ◽  
Author(s):  
A. E. Mayfield ◽  
C. Villari ◽  
J. L. Hamilton ◽  
J. Slye ◽  
W. Langston ◽  
...  

Plant Disease ◽  
2013 ◽  
Vol 97 (9) ◽  
pp. 1248-1248 ◽  
Author(s):  
R. C. Ploetz ◽  
J. Konkol

Gulf licaria, Licaria trianda (Sw.) Kosterm., is a federally endangered member of the Lauraceae plant family in Miami-Dade County, Florida. It was never common in the area, and urban development has extirpated it from most of its former range; as of 2001, fewer than 10 trees remained in a single, remnant habitat in the continental United States, Simpson Park (25°45′31″N, 80°11′46″W) (2). Laurel wilt, caused by the fungus Raffaelea lauricola T. C. Harr., Fraedrich & Aghayeva, has recently devastated members of the Lauraceae in the southeastern United States, most notably redbay, Persea borbonia (1). As R. lauricola and its vector, the redbay ambrosia beetle Xyleborus glabratus, have spread in the region, an increasing number of taxa in this plant family have been affected by this disease (1). In 2012, seedlings of gulf licaria and redbay were obtained from local nurseries; they were grown in 30 liter pots, 1.3 m tall, had stems 3 cm in diameter 20 cm above the soil line, and were maintained with standard watering and fertilization practices. In two pathogenicity experiments on July 6 and September 25, 2012, three plants each of gulf licaria and redbay were inoculated with an isolate of R. lauricola, RL4, as described in previous experiments (3), and two plants each were mock inoculated (water control). RL4 is deposited as CBS 127349 at the Centraalbureau voor Schimmelcultures (CBS Fungal Biodiversity Centre, Utrecht, The Netherlands), and a SSU rDNA sequence for it is deposited in GenBank under Accession No. HM446155. Beginning 2 weeks after inoculation, plants were rated on a weekly basis for the development of external symptoms, on a subjective 1 (no symptoms) to 10 (dead) scale (3). After 5 weeks, inoculated plants of redbay in each experiment (positive control) had died after first developing symptoms of wilt and necrotic foliage that are typical for this disease (1). In contrast, inoculated plants of gulf licaria developed severe symptoms by the time experiments were terminated 6 and 11 weeks after inoculation; chlorosis developed on some of the leaves of all plants and these eventually abscised (mean external severities of 7.3 and 6.5, respectively), but plants did not die. Brown to greyish discoloration of sapwood developed in all inoculated plants, and the pathogen was recovered from symptomatic sapwood on CSMA (3). No symptoms developed on mock inoculated plants and the pathogen was not recovered from them. It is concluded that gulf licaria is susceptible to laurel wilt, but that it is apparently less susceptible than redbay. Whether X. glabratus is attracted to, or will bore into, gulf licaria is not known, but will play a significant role in the extent to which this rare tree is affected by laurel wilt. References: (1) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (2) G. D. Gann et al. Rare Plants of South Florida: Their History, Conservation, and Restoration. Institute for Regional Conservation, Miami, 2002. (3) R. C. Ploetz et al. Plant Pathol. 61:801, 2012.


Plant Disease ◽  
2011 ◽  
Vol 95 (12) ◽  
pp. 1589-1589 ◽  
Author(s):  
R. C. Ploetz ◽  
J. E. Peña ◽  
J. A. Smith ◽  
T. J. Dreaden ◽  
J. H. Crane ◽  
...  

Laurel wilt, caused by Raffaelea lauricola, threatens native and nonnative species in the Lauraceae in the southeastern United States, including the important commercial crop, avocado, Persea americana (2,4). Although the pathogen's vector, Xyleborus glabratus, was detected in Miami-Dade County, FL in January 2010, laurel wilt had not been reported (4). In February 2011, symptoms of the disease were observed on native swampbay, P. palustris, in Miami-Dade County (25°72′N, 80°48′W). Externally, foliage was brown, necrotic, and did not abscise; internally, sapwood was streaked with dark gray-to-bluish discoloration; and, in dead trees, holes of natal galleries of the vector from which columns of frass were attached were evident. On a semiselective medium for R. lauricola, a fungus with the pathogen's phenotype was isolated from symptomatic sapwood. Colonies were slow growing, light cream in color, with dendritic, closely appressed mycelium and often a slimy surface. A representative strain of the fungus was further identified with PCR primers for diagnostic small subunit (SSU) rDNA (1) and its SSU sequence (100% match, GenBank Accession No. JN578863). In each of two experiments, plants of ‘Simmonds’ avocado, the most important cultivar in Florida, were inoculated with three strains of the fungus, as described previously (3). Symptoms of laurel wilt developed in all inoculated plants and the fungus was recovered from each. After aerial and further ground surveys, additional symptomatic swampbay trees, some of which had defoliated, were detected in the vicinity of the original site. Since swampbay defoliates only a year or more after symptoms develop (4), the 2010 detection of X. glabratus may have coincided with an undetected presence of the disease. As of July 2011, a 6-km-diameter disease focus was evident in the area, the southernmost edge of which is 5 km from the nearest commercial avocado orchard. In August 2011, a dooryard avocado tree immediately north of the above focus was affected by laurel wilt, and an SSU sequence confirmed the involvement of R. lauricola (GenBank Accession No. JN613280). The outbreak of laurel wilt in Miami-Dade County represents a 150 km southerly jump in the distribution of this disease in the United States ( http://www.fs.fed.us/r8/foresthealth/laurelwilt/dist_map.shtml ) and is the first time this disease has been found in close proximity to Florida's primary commercial avocado production area. Approximately 98% of the state's commercial avocados, worth nearly $54 million per year, are produced in Miami-Dade County. Since effective fungicidal and insecticidal measures have not been developed for large, fruit-bearing trees, mitigation efforts will focus on the rapid identification and destruction of infected trees (3,4). References: (1) T. J. Dreaden et al. Phytopathology 98:S48, 2008. (2) S. W. Fraedrich et al. Plant Dis. 92:215, 2008. (3) R. C. Ploetz et al. Plant Dis. 95:977, 2011. (4) R. C. Ploetz et al. Recovery Plan for Laurel Wilt of Avocado. National Plant Disease Recovery System, USDA, ARS, 2011.


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