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.     

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.     

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.     

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.     

Pedicularis and Castilleja are natural hosts of Cronartium ribicola in North America: a first report

White pine blister rust disease, caused by the introduced pathogen Cronartium ribicola, has severely disrupted five‐needled pine ecosystems in North America. A 100‐year effort to manage this disease was predicated in part on the premise that the pathogen utilizes only species of Ribes (Grossulariaceae) as alternate hosts on this continent. The current study presents the first conclusive demonstration that some species in the family Orobanchaceae (Pedicularis racemosa and Castilleja miniata) are functioning as alternate hosts in a natural ecosystem of North America. This finding has implications for improving our understanding of epidemiology, pathogen adaptation and host–pathogen interactions within white pine blister rust.     

Resistance of improved Pinus monticola and some other white pines to the blister rust fungus,Cronartium ribicola,of Hokkaido,Japan

Resistant progenies of Pinus monticola, improved in the northwestern USA, and Asian and European white pines were inoculated with the blister rust fungus, Cronartium ribicola, of Hokkaido. No resistance was observed in P. monticola but the other pines remained healthy. This shows that the resistance of white pines to the blister rust fungus is limited to a particular pathogenic race and a different race of the rust fungus exists in Hokkaido.     

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.     

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.     

Endophyte-mediated resistance against white pine blister rust in Pinus monticola

Induced resistance responses, including fungal endophyte-mediated resistance, have been well studied in both agricultural crops and grass systems. Yet, the effect of these processes and symbionts in forest trees is poorly known. Fungal endophytes have been found in all conifer forest systems examined to date and have been hypothesised to be involved in resistance-mediated responses. However, in the absence of functional studies the influence of these endophytes on the extended phenotype of the host plant is unclear. In this study we demonstrate that fungal endophytes from Pinus monticola were effective at increasing survival in host plants against the exotic pathogen Cronartium ribicola, which is responsible for the devastating disease called white pine blister rust. Seedlings previously inoculated with fungal endophytes lived longer than endophyte-free seedlings and also showed some reduction in white pine blister rust disease severity. This endophyte-mediated resistance was found to be effective over time, indicating persistence, and is hypothesised to be a form of induced resistance. Overall, this suggests fungal endophytes may play a determinative role in the structure of biological communities and could provide a useful alternative or ancillary management tool for combating pests and diseases.     

Effects of nursery environment on needle morphology of Pinus monticola Dougl. and implications for tree improvement programs

Statistically significant differences were found in 14 needle traits of western white pine (Pinus monticola Dougl.) seedlings grown from the same seed orchard source in the three nurseries in northern Idaho. Traits with significant variation included needle length and width, number of stomatal rows, number of stomata per row, total stomata per needle, adaxial surface area, stomatal density, major axes of stomata, stomatal shape, stomatal area, stomatal occlusion, epistomatal wax degradation, weight of wax per dry weight of needle, and the contact angles of water droplets placed on adaxial needle surfaces. Wax crystallites on needle surfaces were hollow and tubular and the amount of surface wax appeared to be associated with surface wettability. Our results may have important implications for tree improvement programs that require successful inoculation of nursery-grown seedlings with spores of Cronartium ribicola J. C. Fisch. ex Rabenh. to reliably screen white pines for resistance to blister rust.     

Blister rust in maritime pine

Maritime pine (Pinus pinaster Ait.) seedlings from four stands of central Italy were inoculated with a suspension of 75,000 basidiospores/ml. Significant differences among the stands were found for the following traits: percentages between nonspotted and inoculated seedlings; and percentages between seedlings with spermagonia of Cronartium flaccidum and spotted seedlings. Genetic variation in blister rust resistance among the stands and among the families within a stand confirms the validity of the idea to select the stands which are exposed to heavy rust infection and then within the selected stands to choose trees able to supply the highest possible number of nonspotted seedlings and seedlings with needle spots but without spermagonia.     

Responses of conifer species of the Great Lakes region of North America to inoculation with the pitch canker pathogen Fusarium circinatum

Five conifer species grown in the Great Lakes region of North America were examined for their susceptibility to Fusarium circinatum, (syns. Fusarium subglutinans f. sp. pini and F. moniliforme var. subglutinans), the causal agent of pitch canker. Three‐year‐old (3‐0) seedlings of red (Pinus resinosa), jack (P. banksiana) eastern white (P. strobus), Scots (P. sylvestris) and Austrian (P. nigra) pine were planted in 4 l pots in a greenhouse at Auburn University in November 1998. In April and June 1999, seedlings were inoculated by removing a needle fascicle approximately 5 cm from the terminal bud and placing a drop containing F. circinatum conidia on the wound. Resin production, canker length and seedling mortality were recorded 12 weeks later. Jack, Scots and eastern white pine were the most susceptible with Austrian and red pine more resistant to the fungus. F. circinatum was re‐isolated from 37% to 96% of inoculated seedlings. The susceptibility of jack, Scots and eastern white pine indicates a potential risk to these important species of the region if F. circinatum were to be introduced into the area.     

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.     

Anatomical and cellular responses of Pinus monticola stem tissues to invasion by Cronartium ribicola

White pine blister rust (Cronartium ribicola) causes extensive damage to white pines and their associated ecosystems across North America. The anatomical and cellular characteristics of C. ribicola colonization in Pinus monticola branch and stem tissues were studied as a basis for understanding host tree reactions that may be related to resistance. Samples examined showed typical fusiform swelling and some had produced aecia. The reaction of phloem and xylem tissues was compared with non-infected tissue using light and electron microscopy. Cortical parenchyma and phloem polyphenolic parenchyma cells underwent mitotic division, cell swelling, and ca sixfold greater accumulation of phenolic compounds in colonized vs. control stems. In the cortex and secondary phloem, haustoria were common in parenchymatous cells, and hyphae were abundant in the intercellular spaces, but cell death was rare, unless aecia had ruptured the stem cortex. Hyphae were also common in xylem rays, tracheids and between tracheids. Disease-induced changes in the cambial zone included development of cambium-derived xylem traumatic resin ducts. Results demonstrate that diverse host defence responses were activated in the bark of apparently susceptible trees, but lack of mechanical damage by C. ribicola to the phenol-containing host cells and the resin duct system allowed extensive colonization and development of aecia despite elicitation of these stem defences. Interactions between P. monticola and C. ribicola are discussed and compared with other conifer–fungus pathosystems.     

White pine (Pinus strobus L.) regeneration dynamics at the species’ northern limit of continuous distribution

The abundance of eastern white pine (Pinus strobus L.) has been significantly reduced across its distribution range over the past few centuries. The species’ regeneration dynamics is well documented in the centre of its range, but is poorly understood at the northern limit of continuous distribution. To address this knowledge gap, we quantified natural white pine regeneration in unmanaged mature stands, identified the most important variables influencing it, and evaluated the impact of damaging agents, namely white pine blister rust (Cronartium ribicola J.C. Fisch.), white pine weevil (Pissodes strobi Peck), and herbivory. We also quantified the influence of remnant stands and residual trees on the spatial distribution of regeneration in logged sites. The results reveal continuous but low recruitment in mature stands. The basal area of balsam fir (Abies balsamea (L.) Miller) had a strong negative effect on white pine regeneration. Regeneration was more abundant than expected on moister substrates, including moss, decaying wood and organic matter. White pine regeneration was noted in recently logged areas, where distance from remnant stands had a significant effect on the abundance of white pine regeneration. The northern limit of continuous distribution holds potential for white pine restoration, for example by preserving remnant white pine stands that can provide seed sources for natural regeneration in a shelterwood cut system, or in adjacent clearcut areas. This study illustrates that different management strategies should be used near northern range limits, where effects of site conditions and disturbance agents are different than in the center of a species’ range.     

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.     

Susceptibility of Ribes spp. to pine stem rusts in Finland

Susceptibility of Ribes spp. to three pine stem rusts, Cronartium ribicola, Cronartium flaccidum and Peridermium pini, was investigated by inoculations both in laboratory and in greenhouse conditions, and by observing sporulation on Ribes spp. leaves under natural and artificial inoculum in 16 experiments. Twenty‐seven Ribes spp. cultivars were inoculated in 2000–2004 using 41 sources of C. ribicola aeciospores from a wide geographic range, and six Pinus spp.; 51 sources of C. flaccidum and 11 sources of P. pini from Pinus sylvestris. The results were very similar both after artificial inoculations and observations under natural inoculum in repeated experiments over the years. Cronartium ribicola uredinia and telia developed frequently or moderately on nine Ribes nigrum cultivars but were not present on two cultivars. Sporulation developed on five Ribes rubrum cultivars but was absent on two cultivars. Three Ribes uva‐crispa cultivars were only weakly susceptible to C. ribicola, while all R. alpinum cultivars were resistant to the rust. The other Ribes spp. tested, Ribes niveum, Ribes aureum, Ribes odoratum, Ribes sp. × nigrolaria, Ribes glandulosum and Ribes × culverwellii Jostaberry, were all susceptible to C. ribicola. Cultivars of R. nigrum were more susceptible than those of any other species. No significant variation in virulence of the aeciospores was observed between and among Ribes hosts over the years. Cronartium flaccidum and Peridermium pini did not form any uredinia or telia on Ribes spp. in any of the experiments.     

Blister rust in Romania

In a survey of the incidence of Cronartium ribicola in Romania, this parasite was found to be present all over the country, except in mountain regions. The heaviest attacks occurred in Ribes nigrum and in young plantations of Pinus strobus, whereas old plantations of P. strobus and other pine species were free from blister rust, as were natural populations of Pinus cembra, Ribes alpinum, and R. petraeum in the Carpathians.     

Risk of white pine blister rust to limber pine in Colorado and Wyoming,USA

Cronartium ribicola, the introduced pathogen that causes white pine blister rust (WPBR), continues to spread to additional limber pine populations in the Southern Rocky Mountains of Colorado and Wyoming. Because WPBR can severely impact ecosystems, forecasts of its potential distribution and incidence would be useful to land managers. Site and climate data from long infested study areas in Wyoming were fit with two regression models [logistic and classification and regression trees (CART)] to determine the environmental conditions associated with the distribution of WPBR. These models were then used to map limber pine stands at risk of infestation by C. ribicola throughout Wyoming (where it has long occurred) and Colorado (where it is just becoming established). Although variables representing vegetation and landform could identify infested plots, 1‐km‐scale climate variables for monthly temperature and moisture were better predictors of current WPBR distribution and were available for mapping expected future distribution across the region. Of 280 485 ha where limber pine was projected to occur in Colorado, 41% was forecast by the logistic model to be at risk of infestation, and 53%, by the CART model. Of an estimated 782 229 ha in Wyoming with limber pine, the logistic model projected 61% to be at risk; CART projected 79%. Additional regression models were fit with site and climate data to predict WPBR incidence (per cent of trees infected) and intensification (incidence/age of the oldest canker). Nearly one half of the plot‐to‐plot variation in incidence was explained using environmental variables readily available to land managers. Although mean plot incidence increased over time, mean intensification decreased 50% per decade. This work provides managers with several tools to reduce uncertainty over the expected distribution and incidence of WPBR, but surveillance and monitoring remain prudent activities for supplementing forecasts of WPBR epidemics.     

Reliability of field,greenhouse and cut‐shoot screening procedures for evaluating susceptibility of Scots pine to Melampsora pinitorqua

The reliability of field, greenhouse and cut‐shoot screening procedures for the assessment of the susceptibility of Scots pine (Pinus sylvestris) genotypes to Melampsora pinitorqua, the causal agent of twisting rust, was evaluated. Fourteen genotypes evaluated on half‐sib progenies after natural infection of 2‐year‐old plants showed comparable rank for rust susceptibility with those evaluated after controlled inoculation of 1‐year‐old seedlings in a greenhouse. The dynamic of pycnia production was assessed at 2‐day intervals in cut‐shoot assays under controlled conditions, giving reliable rankings of Scots pine genotypes between years and being significantly correlated with rust susceptibility assessed after natural infection of 2‐year‐old progenies. These results underlined the importance of some critical factors such as inoculum load and host phenology in the reliability of susceptibility evaluations assessed under different experimental conditions. Artificial inoculation of 1‐year‐old seedlings in greenhouse experiments could provide a useful early test for the management of Scots pine breeding programmes and study of inheritance of twisting rust susceptibility. However, in specific investigations the cut‐shoot assay would constitute a reliable laboratory test for studying host–pathogen interactions and the variability in pathogenicity of Melampsora pinitorqua populations.