Field ready: Development of a rapid LAMP-based colorimetric assay for the causal agent of white pine blister rust,Cronartium ribicola

The invasive fungal pathogen Cronartium ribicola causes white pine blister rust which is considered one of the most destructive diseases of five-needle (white) pines in North America. The disease has a life cycle that requires two hosts: white pines and Ribes spp., although other non-Ribes species, including Castilleja and Pedicularis, have been demonstrated as alternate hosts as well. Detection of this disease can be difficult because of the ephemeral nature of sporulation on pine hosts with ambiguity in other symptoms, and the alternate hosts for C. ribicola can also be an alternate host for other pine rust species. We used the previously published C. ribicola genome and species-specific real-time PCR assay to develop a field-ready loop-mediated isothermal amplification (LAMP) specific colorimetric assay for this pathogen. Specificity results across regionally identified pine rust pathogens showed the assay is highly specific to C. ribicola and can detect as little as 40 pg of pathogen DNA. We also developed a simple DNA extraction method that works with several tissue types (bark/phloem, aeciospores, and urediniospores/telia) to prepare the DNA samples for the LAMP assay. The DNA extraction and LAMP assay take ~70 min to complete and require a relatively small investment in equipment. This tool enables quick and efficient detection of white pine blister rust.     

Proactive intervention to sustain high-elevation pine ecosystems threatened by white pine blister rust

Only recently have efforts begun to address how management might prepare currently healthy forests to affect the outcome of invasion by established non-native pests. Cronartium ribicola, the fungus that causes the disease white pine blister rust (WPBR), is among the introductions into North America where containment and eradication have failed; the disease continues to spread. Ecosystem function is impaired by high rust-caused mortality in mature five-needle white pine forests. This paper evaluates five proactive management options to mitigate the development of impacts caused by white pine blister rust in threatened remote high-elevation five-needle pine ecosystems of western North America. They are: reducing pest populations; managing forest composition; improving host vigor; introducing resistant stock with artificial regeneration; and diversifying age class structure to affect the natural selection process for resistance. Proactive intervention to manage and facilitate evolutionary change in the host species may sustain host populations and ecosystem function during pathogen naturalization.     

Histopathology of primary needles and mortality associated with white pine blister rust in resistant and susceptible Pinus strobus

White pine blister rust caused by Cronartium ribicola is a damaging non‐native disease of five‐needled pines in North America. Efforts to control the disease and mitigate damage to date have been only somewhat effective. Recent efforts to improve the health of eastern white pine and reestablish the tree as a dominant species in the North Central United States have focused on identification and propagation of disease‐free eastern white pine (Pinus strobus) growing in areas with a high incidence of blister rust. Many of these selections have been shown to resist infection following artificial inoculation with C. ribicola. In this study, 13 eastern white pine families derived from controlled pollination of selections previously determined to possess putative resistance as well as susceptible selections were inoculated with C. ribicola. Mortality data from inoculation studies show superior survivability in three families with over 60% of seedlings able to survive the 52 week post‐inoculation monitoring period compared to 0–10% survival of the most susceptible families. Primary needles were collected for histological analysis from all inoculated families 4 weeks after inoculation and from selected families 6.5 weeks and 38 weeks after inoculation. Histological observations of infection sites show distinct resistance reactions in the families more likely to survive infection based on mortality data. Analysis of the reactions in susceptible families revealed extensive hyphal colonization of the vascular bundle and adjacent mesophyll cells that appear uninhibited by tree responses. In resistant families, collapsed cells adjacent to infection sites, heavy deposition of phenolic compounds and abnormal cell growth were documented more frequently and appear to play an integral role in the ability of these eastern white pine families to impede growth of C. ribicola in primary needle tissue.     

Breeding eastern white pine: A world-wide perspective

Eastern white pine (Pinus strobus L.) has wide genetic variability and high productivity in plantations in North America, Europe and the Far East. In regions where neither white pine blister rust nor the white pine weevil are a problem, as in the central states of the U.S.A. and the warmer parts of Europe, primary attention is given to selection and breeding for vigor. Estimates for trees up to age 18 indicate that a first-generation genetic gain in volume of over 20% can be obtained from open-pollinated family selection within stands. Volume gains of 50% or more over local genotypes have been realized at ages 15–20 through the use of seed from selected southern Appalachian stands and in some individual tree crosses of eastern white pine with blue (Himalayan) pine. In the cool, humid regions of eastern North America and Europe, where blister rust resistance is important, breeding strategies include cloning of resistant P. strobus individuals, use of blue and Balkan pines in breeding programs, and exploitation of low-level rust resistance in low-risk localities. Eastern white pine losses from blister rust can be avoided in Japan and Korea by good plantation management without genetic selection. In some regions where the white pine weevil is the major problem, western white pine is useful both for hybridization and as an alternative, more weevil-resistant species. Analysis of relative photosynthesis of SO₂- and O₃-fumigated clones may be useful as a method of screening for pollution tolerance in regions where air pollution is likely to intensify. Efficient techniques for cloning by rooting of cuttings are now available. Efforts are under way to develop technology for micropropagation for mass production of superior clones. Breeding strategies for the major P. strobus plantation regions of the world are summarized.     

Incidence and distribution of white pine blister rust in the high‐elevation forests of California

In 2004–2006, a California‐wide survey was conducted to evaluate the incidence and distribution of Cronartium ribicola, cause of white pine blister rust (WPBR), in the high‐elevation white pine forests. WPBR occurrence varied considerably within and between regions, and little to no disease was found in the Basin and Range, eastern Sierra Nevada and Transverse and Peninsular mountain ranges. Field surveys revealed no evidence of rust on Great Basin bristlecone, limber or the southern subspecies of foxtail pine. Rust incidence for western white pine was highest in the North Coast region (42% of trees surveyed), followed by the Klamath (18%) and northern Sierra Nevada (14%). For whitebark pine, WPBR incidence averaged 24% in the northern Sierra Nevada; this was considerably greater than other regions where whitebark pine was found infected. Latitudinal trends in WPBR incidence (i.e. greater in the north) may correspond with earlier introductions of C. ribicola into these regions, reflecting the southward spread of the pathogen since its introduction from the north in the early 1900s and environmental conditions favourable for infection. Distance from inoculum sources and potentially host phenology may also be important factors influencing the distribution of C. ribicola.     

Castilleja and Pedicularis confirmed as telial hosts for Cronartium ribicola in whitebark pine ecosystems of Oregon and Washington

The primary objective of this research was to determine whether native species of Castilleja and Pedicularis are naturally infected by Cronartium ribicola in whitebark pine ecosystems of the Oregon and Washington Cascade Range, USA. Secondary objectives were to monitor the phenology of aecial and telial hosts to determine whether there is sufficient time for C. ribicola to complete its life cycle within high‐elevation stands and to evaluate the variety of susceptible native host species within these genera through field and growth chamber inoculation. These objectives were approached through fieldwork in 2008 and 2009 in whitebark pine ecosystems at Mt. Rainier, Mt. Adams, Mt. Hood, Mt. Bachelor, Tumalo Mtn. and Crater Lake. Forty‐nine observational study plots were established and monitored. Natural C. ribicola infection was detected on 84 Pedicularis racemosa plants and five Castilleja plants (C. applegatei, C. miniata and C. parviflora). Field observation provided evidence that there is sufficient time for C. ribicola to complete its life cycle on hosts within high‐elevation whitebark pine stands. In 2009, 18 field inoculation plots were established at Mt. Rainier and Crater Lake. Field inoculation confirmed the susceptibility of two additional species within these genera, C. arachnoidea and P. bracteosa. All four Castilleja species inoculated in the growth chamber developed infection, with an overall infection incidence of 62% (167 out of 270 plants). The identity of the rust species on field specimens as C. ribicola was verified through PCR and sequencing of the ITS1‐5.8S‐ITS2 region of DNA. Improved understanding of the role of these newly recognized hosts in white pine blister rust epidemiology should be used to prioritize sites for the restoration of ecologically valuable whitebark pine.     

Epicuticular wax content of western white pine is involved in Cronartium ribicola resistance

The introduction of Cronartium ribicola, the causative agent of white pine blister rust, has been devastating for white pines in North America, including western white pine. Among the observed partial resistance responses to white pine blister rust is a mechanism referred to as difficult-to-infect (DI), which is characterized by lower frequency of infection spots on needles and a lower incidence of branch and stem cankers than susceptible host plants. Parents with the DI trait were selected and bred to produce nine full-sib families. Progeny were propagated and cloned using in vitro techniques in controlled environmental conditions. Explants were inoculated with a single strain of C. ribicola and characterized by using a disease assessment index (DAI), which was used to compare DI full-sib families to several other full-sib families (from Idaho) selected for other partial resistance traits. Most DI families had significantly lower DAI scores and higher epicuticular wax content. When the wax was removed from the surface of needles, the DAI of all full-sib families increased. Scanning electron microscopy revealed that stomata in DI full-sib families are mostly occluded by epicuticular wax. The results of this study suggest that the DI resistance mechanism of western white pine is related to needle surface epicuticular wax, which likely hinders the penetration of stomata by fungal hyphae. The occurrence and magnitude of this trait in the breeding population remains unknown, and special assessment technique for large-scale screening will have to be developed.     

A method for estimating white pine blister rust canker age on limber pine in the central Rocky Mountains

Epidemiological studies of white pine blister rust on limber pine require a temporal component to explain variations in incidence of infection and mortality. Unfortunately, it is not known how long the pathogen has been present at various sites in the central Rocky Mountains of North America. Canker age, computed from canker length and average expansion rate, can be used to estimate infestation origin and infection frequency. To investigate relationships between canker lengths and canker ages for limber pine, we collected live white pine blister rust branch and stem cankers from three locations in Wyoming and two locations in Colorado. We quantified relationships between various measures of canker length and an estimate of canker age based on dendrochronological analysis. Total branch canker length was strongly, negatively correlated (r = ?0.79) with the first year of incomplete, annual ring formation (canker age). Mean longitudinal canker expansion rate was 8.4 cm year^?1 for branch and stem cankers where branches distal to the canker were either dead or alive. Annual longitudinal canker expansion, however, was significantly greater on a stem or branch where the portion distal to the canker was alive (11.5 cm year^?1) rather than dead (7.1 cm year^?1). For branches or stems, proximal expansion rate (i.e., toward or down stem) averaged 4.9 cm year^?1. The circumferential canker expansion rate (around branch or stem) was greater for stem cankers (8.3 cm year^?1) than for branch cankers (6.2 cm year^?1). Additional site and host tree covariates did not improve prediction of canker age. Two simple linear equations were developed to estimate a canker age from total length of a canker with the distal portion either alive or dead. An appropriate sample of canker ages can be used to determine how long a limber pine stand has been infested with white pine blister rust and how frequently infections have occurred.     

Regeneration of Rocky Mountain bristlecone pine (Pinus aristata) and limber pine (Pinus flexilis) three decades after stand-replacing fires

Rocky Mountain bristlecone pine (Pinus aristata) and limber pine (Pinus flexilis) are important high-elevation pines of the southern Rockies that are forecast to decline due to the recent spread of white pine blister rust (Cronartium ribicola) into this region. Proactive management strategies to promote the evolution of rust resistance and maintain ecosystem function require an improved understanding of the role of disturbance on the population dynamics of both species and environmental conditions that favor seedling establishment. We examined patterns of bristlecone and limber pine regeneration across the perimeters of three, 29-year-old, high-severity burns in northern, central, and southern Colorado: Ouzel, Badger Mountain, and Maes Creek, respectively. Both species exhibited a very protracted regeneration response to these fires. Bristlecone pine regeneration was concentrated near burn edges and beneath surviving seed sources. This spatial pattern is consistent with limitations incurred by wind-dispersal, also borne out by the low occurrence of seedling clusters. Relative to unburned stands, the absolute abundance of bristlecone pine generally increased only on plots retaining some surviving trees. Limber pine regeneration pattern varied between sites: high in the burn interior at Ouzel, concentrated at burn edges at Badger, and mostly in unburned stands at Maes. Clark’s Nutcracker dispersal of limber pine in each study area was indicated by high seedling distance from possible seed sources and high frequencies of clustered stems. Except at Ouzel, the absolute abundance of limber pine decreased in burns. Across sites, establishment by both species was boosted by nearby nurse objects (rocks, fallen logs, and standing tree trunks), a relationship that extended out at least as far as the closest three such objects, usually found within 50 cm. Fire decreased the frequency of Pedicularis but increased Castilleja and Ribes species (alternate hosts of white pine blister rust), though only one species, R. cereum, was positively associated with either pine species. We conclude that regeneration of bristlecone and limber pine may benefit from natural disturbance or proactive management creating appropriately sized openings and microtopographic structure (e.g., abundant fallen logs); however, beneficial responses may require many decades to be achieved.     

Relative susceptibility to blister rust caused by Cronartium flaccidum of several species of pine

At Florence, Italy, several species of pine were experimentally infected with blister rust. Inoculations were carried out on 3 and 15 months-old seedlings. After antificial and natural inoculation, Brutia, Aleppo, Austrian, Swiss mountain, Maritime and Italian stone pine showed pyenia and aecia. Spotted seedlings of Ponderosa pine showed only mycelium of C. flaccidum in needle and stem tissues. The exotic species seemed to have a very high degree of resistance to blister rust.     

The impact of cronartium flaccidum on the growth of pinus sylvestris

The blister rust of two‐needle pines in Europe is caused by the rust fungus, Cronartium flaccidum (Alb. Schw.) Wint. There are two races of the fungus: One host‐alternating and another pine‐to‐pine race. The latter race is considered to be more common in northern Sweden. The impact of this rust on growth of Scots pine (Pinus sylvestris L.) was investigated in three selected stands in northern Sweden. Radial stem increment was reduced 40–70% by severe attacks and 20–40% by minor attacks. The reduction of stem volume growth was probably greater. The faster growing trees seemd to be more severely attacked. Trees growing on poor soils appeared to lose almost as much growth capacity from a minor attack as from a severe attack.     

A Proposed International Program for Testing White Pine Blister Rust Resistance1

A two-stage, international blister rust resistance test program proposed by the IUFRO Committee on (resistance to) White Pine Blister Rust is described. First, there would be a reevaluation of “naturally” resistant Eurasian white pines from near the original white pine: Cronartium ribicola gene center(s). Next, there would be more sophisticated testing of North American white pines developed by artificial selection and controlled crossing. Key test centers are needed in Siberia, south-central Asia, the Baltic-North Sea countries, and in eastern and western North America. Initial Committee inquiries indicate that 5 of the core cooperators could commence testing in the near future.     

茶藨生柱锈菌致病性分化的初步研究

采用交互接种的方法对华山松疱锈病、红松疱锈病的病原菌茶藨生柱锈菌的致病性分化进行了研究。结果表明:(1)红松疱锈病的病原菌锈孢子只能侵染其本地的转主寄主植物东北茶藨子;(2)华山松疱锈病的病原菌锈孢子能转主侵染多种不同地区的茶藨子植物;(3)所有华山松疱锈病病原菌的锈孢子均不能转主侵染东北茶藨子;(4)初步认定马先蒿属植物不是华山松疱锈病病原菌的转主寄主。     

Recent insights into the pandemic disease butternut canker caused by the invasive pathogen Ophiognomonia clavigignenti‐juglandacearum

In the 25 years following the initial 1967 report of the disease, butternut canker was able to quickly spread throughout the entire range of butternut (Juglans cinerea) in North America, from Minnesota in the upper Midwest to Tennessee in the south and Quebec in the north‐east. The speed of this dispersal is notable as butternut trees do not make up a significant proportion of any single forest type. Instead, they are usually found sparingly in most mixed hardwood forests. In this review, we synthesize the current knowledge of the invasion process of the butternut canker pathogen, Ophiognomonia clavigignenti‐juglandacearum, an invasive fungal pathogen, that as its emergence has spread across North America and is now found wherever butternut naturally occurs. Taxonomic studies have determined that the fungus belongs in the genus Ophiognomonia, which includes a number of saprophytes, endophytes and pathogens of members of the Fagales, rather than the genus Sirococcus, which includes several important pine pathogens. The ability of fungus to be dispersed by rain splash, transported on and in beetle vectors, transmitted by infected seed and successfully colonized several species of Juglans and Carya have all likely contributed to the rapid increase in abundance and severity of disease and tree mortality in the invaded forest ecosystems. Recent genomic and population genetic analyses have determined that there were at least three emergence events. A less virulent strain of the fungus likely has been present in the north eastern United States for over a century, but it was the emergence of a more virulent strain of the fungus in Minnesota and Wisconsin in the 1960s that resulted in range‐wide mortality and pushed butternut to be listed as an endangered species in Canada and a number of states in the United States.     

Relationship between vigour and susceptibility of Austrian pine (Pinus nigra) to blister rust (Cronartium flaccidum)

In a 13-year-old plantation of Austrian pine no apparent relationship between vegetative vigour expressed as height and as stem circumference, and susceptibility to blister rust due to Cronartium flaccidum was found. Under conditions which favour the development of the disease, it is possible to select vigorous and blister rust resistant Austrian pines.     

Phytophthora ramorum is a generalist plant pathogen with differences in virulence between isolates from infectious and dead‐end hosts

Variation in virulence was examined among isolates of Phytophthora ramorum from epidemiologically important or infectious (non‐oak) and transmissive dead‐end (oak) hosts from North America. Twelve isolates representative of the genetic, geographic and host range of P. ramorum in the western United States were inoculated on leaves of Umbellularia californica (bay laurel or bay) and stems of Quercus agrifolia (coast live oak). In spite of extreme genetic similarity among the isolates employed, and even within the same genotype, significant differences in lesion size were measured, suggesting virulence in this pathogen is also controlled by epigenetic factors. A strong positive correlation between lesion size on bay laurel and coast live oak provides experimental evidence P. ramorum is a generalist pathogen that lacks host specificity. Isolates from non‐transmissive oaks were significantly less pathogenic both on oaks and bays than isolates from infectious hosts. These results are essential to further our understanding of the epidemiology and evolutionary potential of this pathogen. A quantitative differential in virulence of isolates from hosts with different epidemiological roles has been described for many animal diseases, but is a novel report for a plant disease.     

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

Elm yellows (EY) is a lethal or decline phytoplasma disease that affects several Ulmus (elm) species and hybrids, which is widespread in North America and Europe. The symptoms vary among the elm species. In those native to North America, main symptoms include epinasty, chlorosis, premature casting of the leaves, yellow to brown discoloration of the phloem in the roots and stem and tree death that usually occurs within 1 or 2 years from the appearance of foliar symptoms. In contrast, affected trees of European and Asian species are primarily characterized by witches’ broom as a specific symptom, do not show phloem discoloration and are less prone to decline. The disease is caused by a relatively genetically homogeneous phytoplasma, the EY agent “Candidatus Phytoplasma ulmi,” a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV‐A. In nature, this pathogen exhibits a high plant host specificity. The elm leafhopper Scaphoideus luteolus is the only confirmed vector of EY phytoplasma in North America, whereas Macropsis mendax has been reported as a natural vector in Northern Italy. However, other insect vectors are likely to be involved in its natural spread. Phytoplasmas of other taxonomic groups or 16SrV subgroups, which are known to infect a wide range of plant hosts, have been identified in naturally infected elm trees. However, the pathological relevance of these “non‐elm” phytoplasmas needs to be confirmed in many cases. Their detection is mainly based on the highly sensitive nested PCR assays, while pathological data are lacking. This study summarizes, within the framework of a single comprehensive review, the current knowledge of EY. Gaps in knowledge of this disease and prospects for future research are also critically discussed.     

Larval performances and life cycle completion of the Siberian moth, <Emphasis Type="Italic">Dendrolimus sibiricus</Emphasis> (Lepidoptera: Lasiocampidae), on potential host plants in Europe: a laboratory study on potted trees

The Siberian moth, Dendrolimus sibiricus, Tschtv. is the most harmful defoliator of coniferous forests in North Asia. The pest has already spread over the Urals and continues moving westwards. Recently, it has been recommended for quarantine in member countries by European and Mediterranean Plant Protection Organization (EPPO). The performances of the pest on coniferous species planted in Europe were assessed on a range of potted trees corresponding to the spectrum of economically important conifers in the EU: European larch Larix decidua, Norway spruce Picea abies, Scots pine Pinus sylvestris, European black pine Pinus nigra, and the North American species: Douglas fir Pseudotsuga menziesii and grand fir Abies grandis. Larvae showed a potential to survive and complete the development on all these host tree species. Favorable hosts were grand fir, European larch, and Douglas fir that allowed higher survival, better larval development, and as a result, yielded heavier pupae and adult moths with higher longevity. Black pine was a poor host but, however, could still support larval and pupal development. Norway spruce and Scots pine had an intermediate behavior. If accidentally introduced to Europe, the Siberian moth may become especially damaging in forest stands predominated by European larch and by the North American firs. Norway spruce and especially the two-needle pines will be less prone to intensive defoliation by this species. The fact that the pest may damage the range of economically important coniferous species should be taken into account in the pest risk assessment for Europe and also for North America where the Siberian moth occurrence is considered likely.     

Distribution and frequency of Cronartium flaccidum on Melampyrum spp. in permanent sample plots in Finland

Abstract

In 2006, the distribution and frequency of pine stem rust Cronartium flaccidum was studied on Melampyrum spp. in permanent sample plots of an EU forest health monitoring program and of a BioSoil program in Finland. The frequency of Melampyrum spp. infected with C. flaccidum was low, but the rust was recorded in new locations of eastern Finland. No uredinia of C. flaccidum were observed in samples collected from over 700 plots. Telia were common on Melampyrum sylvaticum but were occasionally also found on Melampyrum pratense and Melampyrum nemorosum. Melampyrum pratense was the most common species growing in plots on mainly dry sites. Melampyrum sylvaticum was seldom detected, suggesting that alternate hosts are absent from northern Finland or that the current sample plot network is too sparse for effective disease monitoring in the north. No relationship between C. flaccidum, Melampyrum spp. and rust incidence in host trees was observed, which implies that the autoecious Peridermium pini is a more likely pathogen than C. flaccidum locally. Because plots containing M. sylvaticum occur almost solely in southern Finland, the plots with M. sylvaticum reflect the best changes in rust epidemics in that area. In the future, nutrient-rich sites containing M. sylvaticum may serve as a source of rust epidemics. It is unlikely that C. flaccidum will spread to dry sites via M. pratense.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.