scholarly journals Sampling and Detection Strategies for the Pine Pitch Canker (PPC) Disease Pathogen Fusarium circinatum in Europe

Forests ◽  
2019 ◽  
Vol 10 (9) ◽  
pp. 723 ◽  
Author(s):  
Vainio ◽  
Bezos ◽  
Bragança ◽  
Cleary ◽  
Fourie ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Control Measures ◽  
Disease Spread ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Intergenic Spacer Region ◽  
Eu Countries ◽  
Specificity And Sensitivity ◽  
Sampling Protocols ◽  
Reproductive Material

Fusarium circinatum Nirenberg & O’Donnel is listed among the species recommended for regulation as quarantine pests in Europe. Over 60 Pinus species are susceptible to the pathogen and it also causes disease on Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) and species in genera such as Picea and Larix. The European Food Safety Authority considers the probability of new introductions—via contaminated seeds, wood material, soil and growing substrates, natural means and human activities—into the EU very likely. Due to early detection, constant surveillance and control measures, F. circinatum outbreaks have officially been eradicated in Italy and France. However, the global spread of F. circinatum suggests that the pathogen will continue to be encountered in new environments in the future. Therefore, continuous surveillance of reproductive material, nurseries and plantations, prompt control measures and realistic contingency plans will be important in Europe and elsewhere to limit disease spread and the “bridgehead effect”, where new introductions of a tree pathogen become increasingly likely as new environments are invaded, must be considered. Therefore, survey programs already implemented to limit the spread in Europe and that could be helpful for other EU countries are summarized in this review. These surveys include not only countries where pitch canker is present, such as Portugal and Spain, but also several other EU countries where F. circinatum is not present. Sampling protocols for seeds, seedlings, twigs, branches, shoots, soil samples, spore traps and insects from different studies are collated and compiled in this review. Likewise, methodology for morphological and molecular identification is herein presented. These include conventional PCR with a target-specific region located in the intergenic spacer region, as well as several real-time PCR protocols, with different levels of specificity and sensitivity. Finally, the global situation and future perspectives are addressed.

scholarly journals Outbreak of Pitch Canker Caused by Fusarium circinatum on Pinus spp. in Northern Spain

Plant Disease ◽  
2005 ◽  
Vol 89 (9) ◽  
pp. 1015-1015 ◽  
Author(s):  
E. Landeras ◽  
P. García ◽  
Y. Fernández ◽  
M. Braña ◽  
O. Fernández-Alonso ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Morphological Identification ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Northern Spain ◽  
Centraalbureau Voor ◽  
Canker Disease ◽  
Intergenic Spacer Region ◽  
Appl Environ ◽  
Pinus Spp

During the winter of 2003-2004, dieback symptoms were observed on Pinus radiata and P. pinaster in pine nurseries in Asturias (northern Spain). Small groups of affected seedlings appeared randomly distributed throughout the nurseries. The seedlings died rapidly, showing basal needle dieback, stem lesions, resin exudations, and wilting. Isolations from infected material onto potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate and Komada's medium consistently yielded Fusarium sp. cultures. The isolates were transferred to PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The cultures were identified as Fusarium circinatum Nirenberg & O'Donnell (= Fusarium subglutinans Wollenweb. & Reinking), causal agent of pitch canker disease, on basis of the presence of polyphialides and characteristic sterile, coiled, hyphae (2). To further confirm their identity, a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) based on histone H3 gene sequences (4) and a test based on the F. circinatum-specific primers, CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment of the intergenic spacer region of the nuclear ribosomal operon (3), were used. Results obtained with both techniques confirmed the morphological identification of the cultures. A representative culture has been placed in the Centraalbureau voor Schimmelcultures (CBS 117843). The pathogen was isolated only from seedlings of P. radiata and P. pinaster. Other species such as P. nigra, P. sylvestris, and Pseudotsuga menziesii, which were also grown in these nurseries, did not show symptoms. Pathogenicity was confirmed by inoculating 6- to 9-month-old P. radiata and P. pinaster seedlings. Small strips of bark (10 × 1 mm) were cut from the stems and similar sized pieces of PDA colonized by F. circinatum were placed in contact with the open wounds and covered with parafilm. Basal needle dieback was observed 10 days after inoculation that resulted in wilting of the seedlings. F. circinatum was reisolated from the affected stems fulfilling Koch's postulates. Later in the year, symptoms of pitch canker were also observed on 20-year-old P. radiata in one forest plantation in Cantabria (northern Spain). Infected branches and shoots of the trees exudated abundant resin, resulting in resinous cankers. The needles, distal to branch tip infections, wilt, fade to yellow then red, and fall from the tree. Affected trees showed noticeable crown dieback. The isolations from the cankers also yielded F. circinatum cultures that were identified as described above. Although a nonrefereed report appeared in 1998 (1), to our knowledge, this is the first report of F. circinatum on P. radiata and P. pinaster in Spain and in Europe. References: (1) L. D. Dwinell et al. Int. Congr. Plant Pathol. 7th. 3:9, 1998. (2) H. I. Nirenberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004. (4) E. T. Steenkamp et al. Appl. Environ. Microbiol. 65:3401, 1999.

scholarly journals Detection and Quantification of Airborne Conidia of Fusarium circinatum, the Causal Agent of Pine Pitch Canker, from Two California Sites by Using a Real-Time PCR Approach Combined with a Simple Spore Trapping Method

2004 ◽  
Vol 70 (6) ◽  
pp. 3512-3520 ◽  
Author(s):  
Wolfgang Schweigkofler ◽  
Kerry O'Donnell ◽  
Matteo Garbelotto
Keyword(s):  
Real Time ◽  
Filter Paper ◽  
Real Time Pcr ◽  
Fungal Spores ◽  
Seasonal Fluctuation ◽  
Selective Medium ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Intergenic Spacer Region ◽  
Monterey Pine

ABSTRACT Pinus radiata (Monterey pine), a tree native to coastal California and Mexico, is widely planted worldwide for timber production. A major threat to Monterey pine plantations is the fungal disease pine pitch canker, caused by Fusarium circinatum (Hypocreales). We present a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum in the air. The assay is also useful for diagnosing the presence of the pathogen on plants. The test is based on the F. circinatum specific primer pair CIRC1A-CIRC4A, which amplifies a 360-bp DNA fragment in the intergenic spacer region of the nuclear ribosomal operon. Real-time PCR was used to calculate the number of fungal spores present in each reaction mixture by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F. circinatum spores. The filter paper method allows prolonged and more sensitive spore sampling in the field compared to traditional traps using petri dishes filled with selective medium. A field test at two sites in coastal California infested with pine pitch canker was carried out during the summer and fall of 2002. Spore counts were in the range of ca. 1 � 103 to ca. 7 � 105/m2, with the highest spore counts in the fall, suggesting a seasonal fluctuation.

scholarly journals First Report of Pitch Canker on Pines Caused by Fusarium circinatum in Portugal

Plant Disease ◽  
2009 ◽  
Vol 93 (10) ◽  
pp. 1079-1079 ◽  
Author(s):  
H. Bragança ◽  
E. Diogo ◽  
F. Moniz ◽  
P. Amaro
Keyword(s):  
Intergenic Spacer ◽  
Pitch Canker ◽  
Fusarium Circinatum ◽  
Northern Spain ◽  
European Continent ◽  
First Report ◽  
Plant Groups ◽  
Pure Cultures ◽  
Terminal Shoot ◽  
Pine Species

In November of 2007, dieback symptoms (basal needle dieback, wilting, and dieback of terminal shoot) were observed on plant groups of Pinus radiata and P. pinaster in a tree nursery located in Anadia in the central region of Portugal (40°26′N, 08°23′W). Two containers with a total of 112 plants per pine species (with and without symptoms) were collected. Small pieces (5 mm long; two from the roots, stem at the soil level, and the aerial part, totaling six pieces) of 20 symptomatic plants were sterilized with 3% sodium hypochlorite, and isolations were performed on potato dextrose agar (PDA) supplemented with 0.5 mg/ml of streptomycin sulfate. A species of Fusarium was isolated from all infected tissues and pure cultures were obtained by single hyphal tip transfers on PDA and Spezieller Nährstoffarmer agar and incubated at 25°C for 10 days with a 12-h photoperiod. The species was identified as Fusarium circinatum Nirenberg & O'Donnell (= F. subglutinans Wollenweb & Reinking) on the basis of morphological and cultural characteristics (2). They produced white, aerial mycelia, violet pigment, typically three-septate macroconidia with slightly curved walls, single-celled microconidia, and characteristic sterile, coiled hyphae. Microconidia were ovoid or allantoid and born in false heads on aerial polyphialides. The identification was confirmed by PCR with specific primers CIRC1A/CIRC4A, resulting in a 360-bp DNA fragment of the two nuclear ribosomal intergenic spacer regions (3). Pathogenicity tests were performed by inoculating 5- and 9-month-old P. pinaster and P. radiata seedlings, respectively. Plants belonging to P. pinea species (8-month-old), the second most important pine in the country, were also included in the tests. Small strips of bark (10 × 1 mm) were cut from the stems and similar-sized pieces of PDA colonized by two isolates of F. circinatum were placed in contact with the open wounds and covered with Parafilm. Ten seedlings for each pine species, isolate, and control (with sterile PDA) were provided in a total of 90 plants. First symptoms, basal needle and shoot dieback, were observed in P. radiata 8 days after inoculation. One month later, all P. radiata and 70% of the P. pinaster plants were dead. In all P. pinea plants, needles turned red along the main stem, from center to periphery, but only 2% of these plants presented wilting of the terminal shoot after 1 month. No symptoms were observed on control seedlings. F. circinatum was reisolated from symptomatic plants of the three species tested. To our knowledge, this is the first report of F. circinatum in Portugal. Pitch canker, caused by Gibberella circinata (anamorph F. circinatum), is one of the most aggressive pathogens on several pine species in the world (1). In 2005, the fungus was detected in the European continent affecting P. radiata and P. pinaster in northern Spain. References: (1) E. Landeras et al. Plant Dis. 89:1015, 2005. (2) H. I. Niremberg and K. O'Donnell. Mycologia 90:434, 1998. (3) W. Schweigkofler et al. Appl. Environ. Microbiol. 70:3512, 2004.

Characterization of Fusarium spp. isolates by PCR-RFLP analysis of the intergenic spacer region of the rRNA gene (rDNA)

2006 ◽  
Vol 106 (3) ◽  
pp. 297-306 ◽  
Author(s):  
A. Llorens ◽  
M.J. Hinojo ◽  
R. Mateo ◽  
M.T. González-Jaén ◽  
F.M. Valle-Algarra ◽  
...  
Keyword(s):  
Intergenic Spacer ◽  
Rflp Analysis ◽  
Rrna Gene ◽  
Fusarium Spp ◽  
Intergenic Spacer Region ◽  
Pcr Rflp

scholarly journals The Western Progression of Lyme Disease: Infectious and NonclonalBorrelia burgdorferi Sensu LatoPopulations in Grand Forks County, North Dakota

2014 ◽  
Vol 81 (1) ◽  
pp. 48-58 ◽  
Author(s):  
Brandee L. Stone ◽  
Nathan M. Russart ◽  
Robert A. Gaultney ◽  
Angela M. Floden ◽  
Jefferson A. Vaughan ◽  
...  
Keyword(s):  
Lyme Disease ◽  
16S Rrna ◽  
16S Rrna Gene ◽  
North Dakota ◽  
Intergenic Spacer ◽  
Rrna Gene ◽  
Content Type ◽  
Intergenic Spacer Region ◽  
Grand Forks ◽  
The 16S Rrna Gene

ABSTRACTScant attention has been paid to Lyme disease,Borrelia burgdorferi,Ixodes scapularis, or reservoirs in eastern North Dakota despite the fact that it borders high-risk counties in Minnesota. Recent reports ofB. burgdorferiandI. scapularisin North Dakota, however, prompted a more detailed examination. Spirochetes cultured from the hearts of five rodents trapped in Grand Forks County, ND, were identified asB. burgdorferi sensu latothrough sequence analyses of the 16S rRNA gene, the 16S rRNA gene-ileTintergenic spacer region,flaB,ospA,ospC, andp66. OspC typing revealed the presence of groups A, B, E, F, L, and I. Two rodents were concurrently carrying multiple OspC types. Multilocus sequence typing suggested the eastern North Dakota strains are most closely related to those found in neighboring regions of the upper Midwest and Canada. BALB/c mice were infected withB. burgdorferiisolate M3 (OspC group B) by needle inoculation or tick bite. Tibiotarsal joints and ear pinnae were culture positive, andB. burgdorferiM3 was detected by quantitative PCR (qPCR) in the tibiotarsal joints, hearts, and ear pinnae of infected mice. Uninfected larvalI. scapularisticks were able to acquireB. burgdorferiM3 from infected mice; M3 was maintained inI. scapularisduring the molt from larva to nymph; and further, M3 was transmitted from infectedI. scapularisnymphs to naive mice, as evidenced by cultures and qPCR analyses. These results demonstrate that isolate M3 is capable of disseminated infection by both artificial and natural routes of infection. This study confirms the presence of unique (nonclonal) and infectiousB. burgdorferipopulations in eastern North Dakota.

Outbreak ofNeisseria meningitidisC in workers at a large food-processing plant in Brazil: challenges of controlling disease spread to the larger community

2011 ◽  
Vol 140 (5) ◽  
pp. 906-915 ◽  
Author(s):  
B. P. M. ISER ◽  
H. C. A. V. LIMA ◽  
C. De MORAES ◽  
R. P. A. De ALMEIDA ◽  
L. T. WATANABE ◽  
...  
Keyword(s):  
Food Processing ◽  
Purpura Fulminans ◽  
Control Measures ◽  
Disease Spread ◽  
Processing Plant ◽  
Sterile Site ◽  
Matched Case ◽  
Epidemiological Link ◽  
Control Study ◽  
Food Processing Plant

SUMMARYAn outbreak of meningococcal disease (MD) with severe morbidity and mortality was investigated in midwestern Brazil in order to identify control measures. A MD case was defined as isolation ofNeisseria meningitidis, or detection of polysaccharide antigen in a sterile site, or presence of clinical purpura fulminans, or an epidemiological link with a laboratory-confirmed case-patient, between June and August 2008. In 8 out of 16 MD cases studied, serogroup C ST103 complex was identified. Five (31%) cases had neurological findings and five (31%) died. The attack rate was 12 cases/100 000 town residents and 60 cases/100 000 employees in a large local food-processing plant. We conducted a matched case-control study of eight primary laboratory-confirmed cases (1:4). Factors associated with illness in single variable analysis were work at the processing plant [matched odds ratio (mOR) 22, 95% confidence interval (CI) 2·3–207·7,P<0·01], and residing <1 year in Rio Verde (mOR 7, 95% CI 1·11–43·9,P<0·02). Mass vaccination (>10 000 plant employees) stopped propagation in the plant, but not in the larger community.

Genomic fingerprinting ofFrankiamicrosymbionts fromCeanothuscopopulations using repetitive sequences and polymerase chain reactions

1999 ◽  
Vol 77 (9) ◽  
pp. 1220-1230 ◽  
Author(s):  
Soon-Chun Jeong ◽  
David D Myrold
Keyword(s):  
Dna Sequencing ◽  
Dna Analysis ◽  
Repetitive Sequences ◽  
Intergenic Spacer ◽  
Rrna Genes ◽  
23S Rrna ◽  
Genomic Fingerprinting ◽  
Chain Reactions ◽  
Intergenic Spacer Region ◽  
Polymerase Chain

Specificity between Ceanothus species and their microsymbionts, Frankia, were investigated with nodules collected from three geographically separated copopulations of Ceanothus species. Nodules were analyzed using DNA sequencing and repetitive sequence polymerase chain reaction (rep-PCR) techniques. DNA sequencing of the intergenic spacer region between 16S and 23S rRNA genes suggested that Ceanothus-microsymbiotic Frankia are closely related at the intraspecific level. Diversity of the microsymbionts was further analyzed by genomic fingerprinting using repetitive sequences and PCR. A newly designed direct repeat (DR) sequence and a BOX sequence were used as PCR primers after justification that these primers can generate Frankia-specific fingerprints from nodule DNA. Analysis of the nodules using BOX- and DR-PCR showed that Ceanothus-microsymbiotic Frankia exhibited less diversity within each copopulation than among copopulations. These data suggested that geographic separation plays a more important role for divergence of Ceanothus-microsymbiotic Frankia than host plant.Key words: Frankia, Ceanothus, rep-PCR, diversity.

scholarly journals Intraspecific Variation in Cannabis sativa L. Based on Intergenic Spacer Region of Chloroplast DNA.

2000 ◽  
Vol 23 (6) ◽  
pp. 727-730 ◽  
Author(s):  
Mareshige KOHJYOUMA ◽  
I-Jung LEE ◽  
Osamu IIDA ◽  
Kogo KURIHARA ◽  
Kazuya YAMADA ◽  
...  
Keyword(s):  
Chloroplast Dna ◽  
Intraspecific Variation ◽  
Cannabis Sativa ◽  
Intergenic Spacer ◽  
Intergenic Spacer Region
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