Pitch Canker Disease Research Articles

Disruption of trans‐pityol‐mediated attraction by racemic trans‐conophthorin in twig beetle Pityophthorus pubescens

AbstractTwig beetle Pityophthorus pubescens (Coleoptera: Curculionidae: Scolytinae) has been previously associated with the Fusarium circinatum (Hypocreales: Nectriaceae), the pathogen causing pitch canker disease, in P. radiata stands of the Basque Country (Northern Spain). Laboratory and field studies were conducted to evaluate the response of the insect to the racemic mixture of the spiroacetal trans‐7‐methyl‐1,6‐dioxaspiro[4.5]decane, also known as conophthorin. In walking bioassays, addition of 10 and 100 ng of racemic trans‐conophthorin to 1 ng of (±)‐trans‐pityol elicited a negative response in males, whereas females did not show any significant preference. Catches of males in attractant‐baited traps were strongly reduced by racemic trans‐conophthorin at all release rates tested (0.5, 1.0, 1.5 and 2.0 mg/day). In contrast, catches of females were not affected by any of the (±)‐trans‐conophthorin release rate.

Nursery and field experiments to test conifers susceptibility to Pitch Canker disease

Fusarium circinatum is the causal agent of the Pitch Canker disease, which was first detected in Spain in 2004. Not only Pinus species seedlings in nurseries were affected but also Pinus radiata plantations in the forest. Thus, the pathogen has spread out over several pine forests of northern Spain producing substantial economical losses. Consequently, a resistant and viable pine species is required as alternative. The susceptibility of fifteen conifer species was tested in laboratory conditions measuring the germination of seeds and mortality of the emerged seedlings. In addition, a complementary field experiment was also established to evaluate natural infection of different conifers. Chamaecyparis lawsoniana and Sequoiadendron giganteum obtained the best results in the assay developed in the laboratory. On the contrary, the whole of P. radiata seedlings died, confirming that it is the most susceptible species, despite this species (together with Pinus pinaster) showed the highest growth in the control stand of the field assay. Natural infection did not occur during the first two years after plantation. Key words: Susceptibility, conifer, Fusarium circinatum, pitch canker, pathogenicity.

Development and evaluation of a real‐time PCR seed lot screening method for Fusarium circinatum, causal agent of pitch canker disease

SummaryFusarium circinatum is a serious pathogen of Pinus spp. worldwide, causing pitch canker disease. F. circinatum can contaminate seeds both internally and externally and is readily disseminated via contaminated seed. Many countries require screening of pine seeds for F. circinatum before they can be imported. The currently accepted screening method is based on culturing the pathogen on a semi‐selective medium and identifying it using morphological traits. This method is time‐consuming and does not allow for accurate identification of the pathogen to the species level. A bulk DNA extraction and real‐time PCR procedure to screen seeds for the presence of F. circinatum were developed in this study. The real‐time PCR method resulted in the detection of F. circinatum in 5 of 6 commercial seed lots tested and has a lower detection limit of 1 × 10−5 ng of F. circinatum DNA per PCR. The culture‐based method detected Fusarium spp. in four of six of the same seed lots. The real‐time PCR method can be used to screen multiple seed lots in 2 days, whereas the culture‐based method requires a minimum of 1–2 weeks. This new real‐time PCR seed screening method allows for fast, sensitive and accurate screening and can be adapted to handle larger volumes of seeds.

In vitro and in vivo interactions between Trichoderma viride and Fusarium circinatum

Fusarium circinatum, a fungus that causes pitch canker disease, has been present in Europe since at least 2003, when it was detected in northern Spain and found to be producing severe damage in tree nurseries and pine plantations. In this study, we tested a method of biological control of the disease with Trichoderma viride, a fungal species successfully used against many other pathogens. In vitro and in vivo assays were carried out to test the efficacy of this antagonist in controlling F. circinatum. The T. viride isolate exerted a significant effect on the growth of F. circinatum in the in vitro assay, reducing the length of the pathogen colony by half. However, although we tested three different concentrations of the T. viride spore solution, no clear conclusions were obtained with regard to the effects on the Pinus radiata seedlings. To our knowledge, this is the first study carried out with the aim of using Trichoderma spp. to control pitch canker disease.

Screening of Maritime pine (Pinus pinaster) for resistance to Fusarium circinatum, the causal agent of Pitch Canker disease

Pitch canker, caused by the fungus Fusarium circinatum, is an introduced non-native disease on pines in natural and planted stands of Europe. Research has not been conducted to test whether a European native pine species shows genetic variation in susceptibility to this disease. Half-sib families from 39 Pinus pinaster clones and seedlings from one unimproved seed source (control) were evaluated for resistance. Pitch canker resistance was not genetically related to tree growth, but seed weight and germination rates were predictive of time-to-death. Heritabilities and associated genetic gains calculated from the greenhouse experiment were consistent, hi2 = 0.18 and 0.45 for time-to-death and for tree mortality, respectively. These heritabilities are high enough to allow pitch canker to be reduced through appropriate genetic strategies. Results indicated that selection for growth of P. pinaster trees in breeding programs would not necessarily imply an increase of susceptibility to F. circinatum. This research may allow the use of native pine individuals as breeding stock or as sources to produce seeds with moderate levels of tolerance to F. circinatum.

A global climatic risk assessment of pitch canker disease

Pitch canker is a devastating disease of Pinus spp. and Pseudotsuga menziesii (Mirb.) Franco. The pathogen responsible for this disease, Fusarium circinatum Nirenberg & O'Donnell, has spread to many countries within the last three decades. The susceptibility of the widely planted commercial species Pinus radiata D.Don to this pathogen has been of concern to pine forest industries worldwide. Using the process-based distribution program CLIMEX, the global risk of pitch canker establishment was predicted based on a number of climatic variables. The predicted risk of pitch canker establishment by CLIMEX fit well with regions known to have the disease, such as the southeastern United States and Spain. Conversely, the model predicted that the climate in California was not optimal for pitch canker, which fits with the observed lower frequency of natural infections and the strong association with insects in this region. Likewise, Chile, which is known to have F. circinatum in the nurseries but not in the plantation forests, was also predicted to have marginal to suitable climatic conditions for pitch canker establishment. Regions of China, Brazil, Australia, and New Zealand were predicted to have optimal climate conditions for disease establishment. Thus, continued strict quarantine regulations are recommended to prevent the establishment and spread of this pathogen in these countries.

Sensitive Detection of Fusarium circinatum in Pine Seed by Combining an Enrichment Procedure with a Real-Time Polymerase Chain Reaction Using Dual-Labeled Probe Chemistry

Fusarium circinatum is the causal agent of pitch canker disease on numerous Pinus spp. This aggressive fungus may infect pine seed cryptically and, therefore, can easily be spread long distances by the seed trade. F. circinatum has recently been listed as a quarantine organism in numerous countries throughout the world, which prompted the development of a specific and sensitive tool for the detection of this pathogen in conifer seed. A new detection protocol for F. circinatum based on a biological enrichment step followed by a real-time polymerase chain reaction (PCR) assay was developed. Several enrichment protocols were compared and a 72-h incubation of the seed with potato dextrose broth was the most efficient technique to increase F. circinatum biomass before DNA extraction. The relative accuracy, specificity, and sensitivity of the real-time PCR assay was evaluated in comparison with a previously published conventional PCR test on 420 seed DNA extracts. The real-time PCR described here proved to be highly specific and significantly more sensitive than the conventional PCR, and enabled the detection of F. circinatum in samples artificially contaminated with less than 1/1,000 infected seed, as well as in naturally infected samples. Last, in order to routinely check the quality of the seed DNA extracts, a primer-probe combination that targets a highly conserved region within the 18S ribosomal DNA in plants or fungi was successfully developed. This assay allows for quick and reliable detection of F. circinatum in seed, which can help to prevent long-distance spread of the pathogen via contaminated seed lots.

Polymerase chain reaction‐based detection of Fusarium circinatum, the causal agent of pitch canker disease

Pitch canker is a highly damaging disease of Pinus radiata and the New Zealand forest industry is concerned by the potential impact of the disease, should it arrive, in New Zealand. To provide a rapid identification technique for this pathogen, a polymerase chain reaction-based diagnostic method has been developed. The method is able to detect the presence of the pathogen within infected host tissue, as well as infested soil and the reliability of the test has been estimated using Bayesian statistics.

Fungal communities associated with pitch canker disease of Pinus radiata caused by Fusarium circinatum in northern Spain: association with insects and pathogen-saprophyte antagonistic interactions

Association of phloeophagous insects colonizing Pinus radiata in northern Spain with Hypocreales fungi was determined by selective isolation and ITS1-5.8S-ITS2 and histone H3 gene sequencing. The general microbiota consisted of 11 species and was dominated by Fusarium circinatum. Among isolated fungi, Fusarium verticillioides, Fusarium culmorum, Fusarium proliferatum, and Fusarium oxysporum can cause seedling damping-off, whereas Fusarium lateritium is not pathogenic and, thus, was selected together with Penicillium chrysogenum, the second dominant species in aerial wood, for studying their biocontrol capabilities. Replacement series and primary resource capture assays indicated that P. chrysogenum outcompeted F. circinatum. In contrast, in secondary resource capture when F. circinatum appeared later in succession, both P. chrysogenum and F. lateritium completely inhibited its growth; there were no differences between the substrate occupied by both saprophytes. Testing these in vitro generated results in nursery controlled conditions, neither P. chrysogenum nor F. lateritium seed treatments with 450 000 conidia reduced the disease incidence on germination or seedling growth in soil simultaneously infested with the same pathogen inoculum. However, both treatments increased germination percentage in soil with 4500, 45, and 0 F. circinatum conidia inoculum: the respective P. chrysogenum/F. lateritium efficacy ratios were 2.01, 1.75, and 1.40. Both treatments increased seedling height in soil with 4500 and 45 pathogen conidia and reduced necrotic lesions on 1-month-old seedlings infected with 18 000, 180, and 1.8 pitch canker fungus conidia on basal needles. Once promising biocontrol taxa have been identified, further studies about intraspecific variability and efficacy with seed complex natural microbiotas will be essential for developing integrated pest management programs against pitch canker fungus in pine nurseries.

Quantitative Association of Bark Beetles with Pitch Canker Fungus and Effects of Verbenone on Their Semiochemical Communication in Monterey Pine Forests in Northern Spain

The association between 11 species of bark beetles (Coleoptera: Scolytinae) and one weevil (Coleoptera: Entiminae) with the pitch canker fungus, Fusarium circinatum Nirenberg and O'Donnell, was determined by crushing beetles on selective medium and histone H3 gene sequencing. Pityophthorus pubescens (Marsham) (25.00%), Hylurgops palliatus (Gyllenhal) (11.96%), Ips sexdentatus (Börner) (8.57%), Hypothenemus eruditus Westwood (7.89%), Hylastes attenuatus Erichson (7.40%), and Orthotomicus erosus (Wollaston) (2.73%) were found to carry the inoculum. In addition, the root weevil Brachyderes incanus L. (14.28%) had the second highest frequency of occurrence of the fungus. The responses of the insects to a range of verbenone doses were tested in field bioassays using funnel traps. Catches of P. pubescens, a species colonizing branch tips of live trees, were significantly reduced in a log-linear dose-dependent relationship. Catches of I. sexdentatus, an opportunistic species normally attacking fresh dead host material, were also gradually reduced with increasing verbenone dose. Catches of Tomicus piniperda L., O. erosus, Dryocoetes autographus (Ratzeburg), H. eruditus, Xyleborus dryographus (Ratzeburg), Hylastes ater (Paykull), Hylurgus ligniperda (F.), H. attenuatus, and B. incanus were not significantly affected by verbenone. The effects of verbenone were consistent with differences in host-age preference. Semiochemical disruption by verbenone in P. pubescens and I. sexdentatus could represent an integrated pest management strategy for the prevention of the spread of pitch canker disease between different stands. However, several species associated with F. circinatum were unaffected by verbenone, not supporting this compound for prevention of the establishment of potential vectors in Northern Spain.

First Report of Fusarium circinatum, Causal Agent of Pitch Canker Disease, from the Roots of Mature Aleppo Pines in California

A rapid decline of Aleppo pines was observed in the parking lot of the Legoland amusement park in San Diego Co., CA. Although Aleppo pine is a known host for Fusarium circinatum, this is the first report describing resin-soaked sapwood lesions in the roots of mature trees. Previous reports on root infections regarded seedlings. The infestation in this location may have begun with infected roots in planted saplings. The persistent infestation at Legoland represents a significant source of inoculum for this pathogen exotic to California. Accepted for publication 7 June 2006. Published 19 February 2007.

Spatial relationships between nitrogen status and pitch canker disease in slash pine planted adjacent to a poultry operation

Pitch canker disease ( Fusarium circinatum Nirenberg & O'Donnell) causes serious shoot dieback, reduced growth and mortality in pines found in the southern and western USA, and has been linked to nutrient imbalances. Poultry houses with forced-air ventilation systems produce nitrogen (N) emissions. This study analyzed spatial correlations between pitch canker disease and foliar, forest floor, soil, and throughfall N in a slash pine ( Pinus elliottii var. elliottii Engelm.) plantation adjacent to a poultry operation in north Florida, USA. Tissue and throughfall N concentrations were highest near the poultry houses and remained elevated for 400 m. Disease incidence ranged from 57–71% near the poultry houses and was spatially correlated with N levels. Similarly, stem mortality ranged from 41–53% in the most heavily impacted area, and declined to 0–9% at distances greater than 400 m. These results suggest that nutritional processes exacerbate changes in disease susceptibility and expression in slash pine.

Pitch Canker Disease of Pines

ABSTRACT Pitch canker, caused by Fusarium circinatum, is a disease affecting pines in many locations throughout the world. The pathosystem was originally described in the southeastern (SE) United States and was identified in California in 1986. Limited vegetative compatibility group (VCG) diversity in the California population of F. circinatum, relative to the SE United States, suggests the former is a recently established and clonally propagating population. Although the much greater VCG diversity found in the SE United States is suggestive of out-crossing, molecular markers indicate that many vegetatively incompatible isolates are clonally related. This implies that VCG diversity may derive, at least in part, from somatic mutations rather than sexual reproduction. Pitch canker is damaging to many pine species and one at particular risk is Monterey pine (Pinus radiata), which is widely grown in plantations and is highly susceptible to pitch canker. However, some Monterey pines are resistant to pitch canker and some severely diseased trees have been observed to recover. The absence of new infections on these trees reflects the operation of systemic induced resistance, apparently in response to repeated infection by the pitch canker pathogen.

Gibberella circinata

<i>Gibberella circinata</i>

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

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.

Structure and development of pitch canker infected Monterey pine stands at Año Nuevo, California

The sustainability of indigenous Monterey pine ( Pinus radiata D. Don) at Año Nuevo stands in the central coast of California was examined. The foremost management objective in these stands is to establish and maintain stand structures that ensure a sustainable presence of the species in terms of uneven-aged management. The major threats are the proliferation of shade-tolerant tree species and the pitch canker ( Fusarium circinatum) disease. The study was based on data from 17 systematically placed sample plots, measured once, in one stand with a very high degree of variation in stand structure and species composition. The results indicated that the sustainability of Monterey pine is not assured by existing stand structures. Monterey pine regeneration is almost completely lacking. Intensive shading and competition from high stand densities of shade-tolerant broadleaf trees are inhibiting regeneration, and growth of seedlings and saplings. Pitch canker is affecting growth, vigor, and competitive status of Monterey pine, but its progress and long-term impact remain unknown. Single tree or group selection cuttings, combined with treatments that enhance regeneration, are urgently required for the promotion of Monterey pine.

Genetic dissection of fusiform rust and pitch canker disease traits in loblolly pine

Loblolly pine (Pinus taeda L.) exhibits genetic resistance to fusiform rust disease (incited by the biotrophic fungus, Cronartium quercuum f. sp. fusiforme) and pitch canker disease (incited by the necrotrophic fungus, Fusarium circinatum). In this study, a total of 14,015 loblolly pine cuttings from 1,065 clones were screened in controlled greenhouse conditions to identify phenotypes of clones, families, and parents that guide a genetic dissection of disease traits associated with pitch canker and fusiform rust. A total of 23,373 phenotypic data points were collected for lesion length (pitch canker) and gall score, gall length, and gall width (fusiform rust). We verified heritable fusiform rust and pitch canker traits and calculated parental, clonal, and full-sib family rankings for both diseases. Genetic correlations revealed that traits associated with fusiform rust are genetically distinct from one another, and that the genetic mechanisms underlying pitch canker and fusiform rust resistance are independent. The disease phenotyping described here is a critical step towards identifying specific loci and alleles associated with fusiform rust and pitch canker resistance.

Variation in susceptibility to pitch canker disease, caused by Fusarium circinatum, in native stands of Pinus muricata

Two hundred Pinus muricata trees, located within a native forest near Monterey, California, were inoculated with the pitch canker pathogen. Treated branches were removed 10–13 weeks following the initial inoculation, and the lengths of the lesions that developed at each of the inoculation sites were measured. Results indicated that bishop pine exhibits a wide range of variation in susceptibility to pitch canker disease. Of the trees that received only one inoculation, 27% showed almost no lesion development, indicating that they were relatively resistant to the pathogen, while others had significantly longer lesions and thus were more susceptible. Clonal propagation and seed collection from resistant individuals may offer useful strategies for disease management in the future.

Sequence characterized amplified polymorphic markers for the pitch canker pathogen, Fusarium circinatum

Abstract Fusarium circinatum is the causal agent of pitch canker disease of pines. This pathogen is thought to have originated in Central America and currently poses a serious threat to commercial pine plantations in many areas of the world. In this study, polymorphic molecular markers were developed for F. circinatum using the internal short sequence repeats‐polymerase chain reaction (ISSR‐PCR) technique. Nine sequence characterized amplified polymorphic markers were developed. Thirty‐two putative alleles were observed among 103 F. circinatum isolates from different geographical areas using the nine polymorphic markers. These sequence characterized amplified polymorphic markers can be used as genetic tools in populations studies of F. circinatum.

The Epidemiology of Pitch Canker of Monterey Pine in California

Abstract The progression of pitch canker disease, caused by Fusarium circinatum, was followed over 4 yr (1992–1996) in urban stands of Monterey pine, Pinus radiata, with initially zero, light, moderate, and severe disease intensity. The disease progressed from a few to many branch tip symptoms, followed by the appearance of cankers on the main stem. Symptomatic branch tips at the start of the study were predictive of tree mortality or removal after 4 yr. Trees with canopy dieback and stem cankers were often attacked by the red turpentine beetle, Dendroctonus valens. Among initially disease-free trees, disease progression and final severity were greater in plots with initially severe and moderate disease intensity. Apparent infection rates did not differ between plots with different initial disease intensities. Over the 4 yr of the study, 2% of trees in the five plots with most severe disease never exhibited disease symptoms and thus may be resistant to pitch canker. In addition, 7% of symptomatic trees in these plots never had more than two symptomatic branch tips, which may reflect a moderate level of resistance. FOR. SCI. 48(4):694–700.