Testing of selected South African <Emphasis Type="Italic">Pinus</Emphasis> hybrids and families for tolerance to the pitch canker pathogen, <Emphasis Type="Italic">Fusarium circinatum</Emphasis>

Plantations of Pinus spp. constitute approximately 50% of the South African forestry industry. The first aim of this study was to develop a reliable inoculation technique to screen Pinus spp., for tolerance to infection by F. circinatum, which threatens pine forestry in South Africa. Inoculation of branches was compared with stem inoculations and we considered the number of branches or trees required to obtain statistically significant results. Furthermore, variation in the susceptibility of some Pinus families, clones and hybrids was considered. Results showed that branch inoculations were closely correlated with those from stem inoculations, and that it is important to consider branch and stem diameters when assessing susceptibility of trees. Subsequent trials using branch inoculations showed significant differences in F. circinatum tolerance amongst a range of pine species and hybrids of potential interest to forestry in South Africa. Significant differences in susceptibility were also found among clones of two P. radiata families. The most tolerant trees were P. elliottii × caribaea and P. patula × oocarpa hybrids, while the most susceptible species were P. patula, P. greggii and hybrids of these two. This is the first trial considering the susceptibility of Pinus hybrids, Pinus clones and some P. patula provenances, and the results indicate excellent potential for breeding for tolerance to pitch canker in South Africa. Application The accurate selection of disease tolerant planting stock for the South African forestry industry is crucially important for the continued sustainability of this important industry. The work described here provides valuable information on an artificial inoculation technique that will assist the industry in screening trees for tolerance to the pitch canker fungus, F. circinatum. It also provides some indication of the relative susceptibility of a number of Pinus spp., hybrids and families currently being evaluated in the country.     

Selection of Pinus spp. in South Africa for tolerance to infection by the pitch canker fungus

The increasing threats from pests and diseases demand that the South African forest industry explores options to deploy alternative pine species in plantation development. This is especially true for species, such as Pinus patula Schiede and Deppe ex Schltdl. and Cham., which are highly susceptible to the pitch canker fungus Fusarium circinatum. Losses due to F. circinatum have been confined mostly to nurseries and at field establishment resulting in a significant cost to the industry. Although, the fungus has not as yet resulted in stem and branch infections on established P. patula in South Africa, it has caused pitch canker on other, more susceptible species such as P. radiata D. Don., and P. greggii Engelm. ex Parl. As alternatives to P. patula, on the warmer and cooler sites in South Africa, families of P. elliottii Engelm var. elliottii, P. tecunumanii (Schw.) Eguiluz and Perry, P. maximinoi H. E. Moore and P. pseudostrobus Lindl. were screened for tolerance to infection by F. circinatum in greenhouse studies. Seedlings were wounded and inoculated with spores of F. circinatum. Lesion development following inoculation was used to differentiate the levels of tolerance between families. The results showed that P. maximinoi, P. pseudostrobus, and the low elevation variety of P. tecunumanii are highly tolerant to infection with very little family variation. The narrow sense heritability estimates for these species were less than 0.06. In contrast, P. elliottii showed good tolerance with some family variation and a heritability of 0.22, while the high elevation source of P. tecunumanii showed a high degree of family variation and a heritability of 0.59. These results provide the industry with valuable information on pine species tolerant to F. circinatum that could be used as alternatives to P. patula in South Africa.     

Frost tolerance of various Pinus pure species and hybrids

Pinus species are widely planted by the South African forestry industry and are utilised for pulp, paper and saw timber products. Historically, Pinus patula Schiede ex Schltdl. et Cham. was the most widely planted commercial species in the summer rainfall area, but has come under severe threat due to the fungus Fusarium circinatum. Fusarium circinatum causes mortality in nurseries and in-field after establishment. Other Pinus species, such as P. tecunumanii F.Schwerdtf. ex Eguiluz et J.P.Perry and P. oocarpa Schiede ex Schltdl., have been crossed with P. patula to increase tolerance to F. circinatum and these hybrids have largely overcome the post-planting mortality problem. However, these hybrid-partner species are more prone to frost damage. This study reviewed laboratory screening techniques to assess the frost tolerance level of a range of Pinus pure species and hybrids. In vitro screening was done with the electrolyte leakage and whole-plant freezing techniques. Seedlings and rooted cuttings from a range of genotypes, supplied by Sappi, were tested in vitro at different target temperatures (?3, ?6, ?9 and ?12 °C) to determine their relative frost tolerance. These genotypes included a range of Pinus pure species, four interspecific hybrids (P. patula × P. tecunumanii low elevation [LE], P. patula × P. tecunumanii high elevation [HE], P. elliottii Engelm. × P. caribaea Morelet), and a three-way cross (P. patula × (P. patula × P. oocarpa)). Results from this study indicated that the electrolyte leakage technique was a reliable method to determine frost tolerance under laboratory conditions, with similar pure species rankings as experienced under field conditions. The interspecific hybrids of P. patula × P. tecunumanii LE and HE ranked intermediate between the parental species and the P. patula × P. tecunumanii HE hybrid was more frost tolerant than the P. patula × P. tecunumanii LE hybrid.     

Future outlook for Pinus patula in South Africa in the presence of the pitch canker fungus (Fusarium circinatum)

Approximately 50% of the area planted to softwood trees in South Africa has been established with Pinus patula, making it the most important pine species in the country. More effort has gone into developing this species for improved growth, tree form and wood properties than with any other species. This substantial investment has been threatened in the last 10 years by the pitch canker fungus, Fusarium circinatum. The fungus infects and contaminates nursery plants and, once transferred to the field, causes severe mortality of young trees in the first year after establishment. Although nurserymen have some control of the disease, it is recognised that the best long-term solution to mitigate damage because of F. circinatum infection is to identify tolerant species, clones and hybrids for deployment in plantations in the future. Research has shown that alternative species such as P. tecunumanii, P. maximinoi and P. elliottii are suitable for warm sites. Pine hybrids, particularly between P. patula and the high-elevation sources of P. tecunumanii, appear to be a suitable replacement on subtemperate and temperate sites. Although these alternative species and hybrids are more sensitive to subfreezing temperatures than P. patula, their planting range can be increased by including cold tolerance as a selection criterion. Future breeding efforts will most certainly focus on improving the tolerance of pure P. patula to F. circinatum, which can be achieved by identifying specific family crosses and tolerant clones. The commercial deployment of disease-tolerant control-pollinated P. patula and hybrid families will most likely be established as rooted cuttings, which requires more advanced propagation technology. In the long term, new seed orchards comprised of P. patula clones tolerant to F. circinatum could be used to produce seed for seedling production.     

The tolerance of Pinus patula × Pinus tecunumanii, and other pine hybrids, to Fusarium circinatum in greenhouse trials

The field survival of Pinus patula seedlings in South Africa is frequently below acceptable standards. From numerous studies it has been determined that this is largely due to the pitch canker fungus, Fusarium circinatum. Other commercial pines, such as P. elliottii and P. taeda, show good tolerance to this pathogen and better survival, but have inferior wood properties and do not grow as well as P. patula on many sites in the summer rainfall regions of South Africa. There is, thus, an urgent need to improve the tolerance of P. patula to F. circinatum. Operational experience indicates that when P. patula is hybridized with tolerant species, such as P. tecunumanii and P. oocarpa, survival is greatly improved on the warmer sites of South Africa. Field studies on young trees suggest that this is due to the improved tolerance of these hybrids to F. circinatum. In order to test the tolerance of a number of pine hybrids, the pure species representing the hybrid parents, as well as individual families of P. patula × P. tecunumanii, a series of greenhouse screening trials were conducted during 2008 and 2009. The results indicated that species range in tolerance and hybrids, between P. patula and these species, are intermediate in tolerance to F. circinatum. Within P. patula × P. tecunumanii, large family variation exists when pollen from the high elevation source of P. tecunumanii is used. The results of these studies illustrate the importance of developing pine hybrid breeding programs to overcome the susceptibility of our pure species to pathogens such as F. circinatum.     

Growth and dynamic modulus of elasticity of Pinus patula × Pinus tecunumanii hybrids in Mpumalanga,South Africa

Field establishment of South Africa’s most important commercial pine species, Pinus patula, is severely hampered by the pitch canker fungus, Fusarium circinatum. Importantly, hybrids between P. patula and other pine species tolerant to the pitch canker fungus, such as P. tecunumanii and P. oocarpa, have been identified as an alternative planting stock. In this study, variation in tree volume and dynamic modulus of elasticity (MOE_dym) of the P. patula × P. tecunumanii (low- and high-elevation [LE and HE] ecotypes) hybrid was compared with the P. elliottii × P. caribaea hybrid, and the pure species P. tecunumanii (LE) and P. patula. The MOE_dym was assessed using the Fakkop TreeSonic microsecond instrument across three sites. The results of the study showed that P. patula × P. tecunumanii LE performed significantly better than P. patula × P. tecunumanii HE for volume and MOE_dym, which in turn was significantly better than P. patula. The MOE_dym and tree growth decreased with an increase in elevation. There was significant taxon × site interaction for volume and MOE_dym. The results of these trials suggested that P. patula × P. tecunumanii LE is a suitable alternative to P. patula in the Sabie region of Mpumalanga in South Africa on frost-free sites, in terms of the traits that were assessed.     

Tolerance of Pinus patula full-sib families to Fusarium circinatum in a greenhouse study

The pitch canker fungus, Fusarium circinatum, has caused large-scale mortality of young Pinus patula Schiede and Deppe ex Schltdl. and Cham. seedlings in nurseries in South Africa since 1990. Diseased seedlings have been inadvertently carried to the field, which in turn have died and has reduced stocking below an acceptable level. Tree breeders have suggested that the only long-term solution to limit infection by this pathogen is to identify and deploy tolerant P. patula families. A commonly used technique to identify tolerant clones is to artificially inoculate open-pollinated progeny from orchard clones with F. circinatum under greenhouse conditions. In these trials, large variation in tolerance to the pathogen among seedlings within open-pollinated families was observed and this could be influenced by the pollen parent. Therefore, identifying individual full-sib families, where both parents are known, should improve the identification of tolerant families, which can then be repeated. In this study, cuttings from control-pollinated P. patula seedling hedges were inoculated with F. circinatum in a greenhouse. The results showed large family variation where some of the full-sib families were similar in tolerance to P. elliottii Engelm. var. elliottii seedlings. Therefore, it is recommended that breeders focus on identifying specific family combinations that are more tolerant to F. circinatum.     

Variation in pitch canker resistance among provenances of <Emphasis Type="Italic">Pinus patula</Emphasis> and <Emphasis Type="Italic">Pinus tecunumanii</Emphasis> from Mexico and Central America

Pinus patula and high-elevation (HE) sources of P. tecunumanii exhibit intermediate levels of resistance to pitch canker (Fusarium circinatum), compared to extremely resistant species such as P. oocarpa, and extremely susceptible species such as P. radiata. Seedlings from 20 P. patula provenances and 15 HE P. tecunumanii provenances were artificially inoculated with the pitch canker fungus at 21 and 12 weeks of age, respectively, and assessed for resistance 12–20 weeks later. There was important provenance variation in pitch canker resistance for both species. The 20-week LiveStem percentage ranged from 70.3% to 43.6% among the P. patula provenances and 59.6% to 11.7% among HE P. tecunumanii provenances. There was a geographic pattern to the provenance variation, and in both species, low altitude sources demonstrated more resistance than those from high elevation. Provenance variation in pitch canker resistance could be useful when making selection and breeding decisions with these species.     

Resistance levels of Spanish conifers against Fusarium circinatum and Diplodia pinea

Pitch canker, caused by Fusarium circinatum, and Diplodia shoot blight, caused by Diplodia pinea, are both damaging to pines (Pinus spp.) grown in plantations throughout the world, including Spain. To assess the potential for interspecific differences in susceptibility to contribute to the management of pitch canker and Diplodia shoot blight in the Atlantic region of Spain, the present study was undertaken to characterize the susceptibility of six pine species (P. sylvestris, P. nigra, P. pinaster, P. radiata, P. halepensis and P. pinea) and Douglas‐fir (Pseudotsuga menziesii) to F. circinatum and D. pinea. Based on inoculations of 2‐year‐old trees, Ps. menziesii, P. pinea and P. nigra were the most resistant to F. circinatum, with lesion lengths ranging from 3.7 to 21.5 mm, 2.2 to 12.6 mm and 2.8 to 30.9 mm, respectively. At the other extreme, Pinus radiata was the most susceptible, sustaining lesions that ranged from 8.5 to 74.8 mm in length. Pinus sylvestris, P. pinaster and P. halepensis showed an intermediate response to F. circinatum. Broadly similar results were observed in inoculations with D. pinea, with Ps. menziesii being relatively resistant and P. radiata being highly susceptible. Consistent with these results, field surveys revealed no pitch canker in stands of Ps. menziesii and low severity of Diplodia shoot blight, whereas P. radiata was severely affected by both diseases. Our findings suggest that selection of appropriate species can greatly reduce the risk of damage from two important canker diseases affecting pine plantations in the Atlantic region of Spain. Furthermore, intraspecific variation in susceptibility implies that selection may allow for the enhancement of resistance in otherwise susceptible species.     

Variation in susceptibility of six pine species and hybrids to pitch canker caused by Fusarium circinatum

Six pine species or hybrids were tested for susceptibility to pitch canker caused by Fusarium  circinatum. Pinus  densiflora, Pinus  thunbergii, Pinus  x rigitaeda (Pinus  rigida × Pinus  taeda), P. rigida × P. x rigitaeda, Pinus  echinata and Pinus  virginiana were inoculated with three spore loads (50, 500 and 5000 per tree) of F. circinatum. External symptoms, lesion length, and the frequency of reisolation of the fungus were investigated. External symptoms were greatest in P. echinata, followed by P. virginiana, however, P. densiflora was not susceptible to F. circinatum. Based on mean lesion lengths, the six pine species or hybrids differed significantly (p < 0.01) in susceptibility to pitch canker. Pinus  echinata sustained the longest lesions, whereas P. densiflora sustained the shortest lesions. The effect of inoculum density was not significant among three spore treatments within species (p = 0.17), although lesion length was slightly greater at higher spore loads over all pine species. The fungus was reisolated from inoculated stems of all pine species tested, even on trees showing little or no damage from the disease. Additional studies are needed to further explore the basis for resistance to pitch canker.     

Sources of Diplodia pinea endophytic infections in Pinus patula and P. radiata seedlings in South Africa

Diplodia pinea, an opportunistic and latent pathogen, can significantly affect Pinus productivity worldwide. Despite being studied in South Africa for almost 100 years, the source of D. pinea inoculum responsible for seedling infection is unknown. The aim of this study was to determine the role of seed in vertical transmission of D. pinea and to investigate sources of inoculum leading to horizontal transmission to pine seedlings. Surface‐disinfected seeds were inoculated with spore and mycelium suspensions of D. pinea to determine its effect on germination. In addition, isolation of the fungus was performed from surface‐disinfected seeds, asymptomatic seedlings collected from nurseries, plantations where pines naturally regenerate and recently established fields, to assess transmission and incidence of endophytic D. pinea infections. Inoculation of seeds with D. pinea spore suspensions affected speed and rate of germination. The fungus was isolated from surface‐disinfected seeds in only a few instances (2–3%) and was not found in healthy seedlings collected from greenhouses and nurseries, suggesting that vertical transmission of the fungus does not occur or is rare. In contrast, D. pinea was isolated from 40% of seedlings obtained from the understory of mature P. patula trees showing that horizontal transmission from mature to young trees sustains the D. pinea inoculum in South African pine plantations.     

Susceptibility of provenances and families of Pinus maximinoi and Pinus tecunumanii to frost in South Africa

The future of South Africa’s most important pine species, Pinus patula, is threatened by the pitch canker fungus, Fusarium circinatum. Pinus maximinoi and P. tecunumanii represent two subtropical species that provide an alternative to planting P. patula on the warmer sites of South Africa. Extending the planting range of P. tecunumanii and P. maximinoi to include higher and colder altitude sites will reduce the area planted to P. patula and the risk of F. circinatum. During 2007 progeny trials of P. tecunumanii and P. maximinoi were planted on a sub-tropical and sub-temperate site. Shortly after the establishment of these trials, unusually cold weather conditions were experienced across South Africa (?3°C at the sub-temperate site) resulting in severe mortality. This provided the opportunity to assess the variation in survival as a measure of frost tolerance within these two species to determine whether it could be improved upon through selection. Results indicated that the variation in survival was under genetic control in P. tecunumanii (h _(0,1) ²  = 0.16, h _L ²  = 0.27) and P. maximinoi (h _(0,1) ²  = 0.11, h _L ²  = 0.23) at the sub-temperate site. Correlations in provenance ranking for survival across sites were high for both species. Moderate correlations in family survival for P. tecunumanii (r = 0.52) were found at the two sites. Improvements in cold tolerance can thus be made in both species extending their planting range to include greater areas planted to P. patula thereby limiting the risk of F. circinatum.     

Alternative pine hybrids and species to Pinus patula and P. radiata in South Africa and Swaziland

Through the collaborative efforts of companies affiliated with the International Program for Tree Improvement and Conservation (Camcore), a number of pine hybrids have been produced over the last decade. Many of these have been planted in trials across southern Africa that broadly represent winter and summer rainfall areas, with the latter ranging from warm to cold temperate sites. The five-year survival and growth of the hybrids and other pines in 12 of these trials were compared with Pinus radiata in the winter rainfall, and P. patula in the summer rainfall, regions where these species have been planted extensively. Except for the highest altitude site, where freezing conditions are common, the survival of most hybrids and tropical pines was better than P. patula or P. radiata. This was, in part, attributed to their improved tolerance to the pitch canker fungus, Fusarium circinatum, which was present in the nursery at the time of planting. In the winter rainfall area, the P. elliottii × P. caribaea hybrid, P. maximinoi and, surprisingly, the P. patula hybrids performed well. In the summer rainfall regions, hybrids with tropical parents such as P. caribaea, P. oocarpa and P. tecunumanii were more productive in the subtropical/warm temperate zone and, with increasing elevation, those hybrids crossed with P. patula performed relatively better. The P. patula × P. tecunumanii hybrid, particularly when crossed with low-elevation P. tecunumanii, performed exceptionally across most sites.     

Evaluation of Pinus radiata seed treatments to control Fusarium circinatum: effects on seed emergence and disease incidence

In this study, the effects of hot water (HWT), hydrogen peroxide and fungicides on the incidence of Fusarium circinatum on artificially inoculated Pinus radiata seeds were evaluated. Fifteen commercial fungicide formulations were screened in vitro for inhibitory activity on mycelial growth and conidial germination of F. circinatum. With half‐maximal effective concentration (EC₅₀) lower than 0.5 ppm, fluazinam, imazalil and tebuconazole were the most effective fungicides on mycelial growth, while captan, mancozeb or pyraclostrobin were the most effective (EC₅₀ < 0.3 ppm) on conidial germination. Based on the results obtained, imazalil, fluazinam, mancozeb and pyraclostrobin were selected for further testing. The effects of HWT, hydrogen peroxide and fungicide treatments on seed emergence and the incidence of F. circinatum were assessed. Seed treatments with fungicides prior to sowing were less effective and inconsistent in reducing the incidence of F. circinatum on seedlings. In contrast, hot water and hydrogen peroxide treatments significantly reduced F. circinatum contamination on P. radiata seeds with an overall disease incidence lower than 0.8% on seedlings. Furthermore, subsequent application of fungicides on seedlings did not improve the effectiveness of HWT. These results, therefore, suggest that hot water is a better alternative to hydrogen peroxide and fungicides as Pinus seed treatment against F. circinatum and could easily be implemented as standard in commercial nurseries to control the spread of the pitch canker disease.     

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.     

Genetic variation in survival,growth, and stem form of Pinus leiophylla in Brazil and South Africa and provenance resistance to pitch canker

Pinus leiophylla is a relatively common pine that occurs in the mountains of western and central Mexico. Between 1987 and 1990, Camcore, North Carolina State University, sampled 11 populations and 309 mother trees of the species to determine patterns of genetic variation in survival, growth and stem form. Fifteen provenance/progeny trials were established in southern Brazil and South Africa across a wide range of sites and assessed at 3, 5 and 8 years of age for height, diameter and stem form. Results indicated that average productivity ranged from 5 to 19m³ ha^?1 y^?1 depending on the site and that performance was not as competitive as P. patula (South Africa) and P. taeda (Brazil) controls. Provenances from central Mexico (Michoacán) were statistically better in productivity than those from the southern part of the country (Oaxaca), which in turn were superior to those from the northern part of the country (Durango). However, provenances from northern Mexico exhibited superior survival to seed sources from other locations in Mexico when grown on cold sites (winter minimums –10 to 5°C) in South Africa and superior stem form across all locations. Seedlings from the original 11 provenances were screened for resistance to the pitch canker fungus (PCF; Fusarium circinatum). Results indicated that generally P. leiophylla is as susceptible to PCF as P. patula (stemkill = 85%) with the exception of two fast-growing, moderately resistant populations from Michoacán, La Pinalosa (stemkill = 44%) and Ario de Rosales (stemkill = 73%). The future potential of P. leiophylla might be in hybrid combination with P. patula in the seasonally dry areas of southern Africa where fires are common and pitch canker is a problem.     

Identification of the Armillaria root rot pathogen in Ethiopian plantations

Armillaria root rot is a well‐known disease on a wide range of plants, world‐wide. In Ethiopia, the disease has previously been reported on Pinus spp., Coffea arabica and on various native hardwoods. The causal agent of the disease has been attributed to Armillaria mellea, a species now known to represent a complex of many different taxa. The aim of this study was to determine the extent of Armillaria root rot and the identity of the Armillaria sp. in Ethiopian plantations. As part of a plantation disease survey in 2000 and 2001, samples were collected in plantations at and around Munessa Shashemene, Wondo Genet, Jima, Mizan and Bedele, in south and south‐western Ethiopia. Basidiocarps were collected and their morphology studied. Morphological identification was confirmed by sequencing the intergenic spacer (IGS‐1) region of the ribosomal rRNA operon and comparing data with published sequences of Armillaria spp. Armillaria isolates were collected from Acacia abyssinica, Pinus patula, Cedrela odorata and Cordia alliodora trees. Sporocarps were found on stumps of native Juniperus excelsa. Basidiocarp morphology and sequence data suggested that the fungus in Ethiopia is similar to that causing disease of Pinus spp. in South Africa and previously identified as A. fuscipes. This identification was confirmed for all isolates, based on sequence data. Armillaria fuscipes is known to be common in southern Africa. Its widespread occurrence in Ethiopia suggests that it is also the major cause of Armillaria root rot in that country.     

Assessment of molecular detection of Fusarium circinatum in insects and passive spore traps in Pinus radiata plantations

Fusarium circinatum is the causal agent of pitch canker, a destructive disease that threatens natural and planted pine forests around the world. Although pitch canker has caused problems in Spain and Portugal, concerning Europe as a whole, the fungus is not established across the pine distribution area. Its dispersion by wind and/or insect vectors could nevertheless play a role in the colonization of currently uninfected stands. It is therefore crucial to develop monitoring tools for its detection. To this end, we assessed the molecular detection of the pathogen in environmental samples of bark beetles and passive spore traps, collected in two infected Pinus radiata plantations in Basque country, Spain. The spread pattern of F. circinatum was assessed by an experimental design that included insect and spore traps installed at the centre, at the edge and outside the plots. Our results showed that F. circinatum was detected in both types of samples, at almost all collection dates. In both type of samples, positive detections were mainly found at the centre of the plots, a lower proportion at the edge, and very few outside. This suggests that long‐distance dispersion of Fusarium circinatum does not rely on wind spore dispersal neither on insect flight. Our study also shows that molecular methods are a powerful tool to monitor the pathogen in environmental samples.     

Environmentally induced changes in the distribution of disease susceptibility phenotypes in Pinus radiata

Pinus radiata (Monterey pine) is highly susceptible to Fusarium circinatum, the cause of pitch canker, but heritable variation in resistance to this disease has been documented in P. radiata. In this study, the distribution of susceptibility phenotypes (=lesion lengths) was assessed by inoculating P. radiata trees with a spore suspension of F. circinatum. The results show the distribution of susceptibility phenotypes in naïve seedlings to be normally distributed, both in a breeding population and in a population established from seed collected in a native forest. This pattern aligns with expectations for a quantitatively inherited trait. However, after 2.5 years of growth in a forest, variation in susceptibility is no longer normally distributed but rather is positively skewed. Thus, exposure to the forest environment results in a distribution that is enriched for relatively resistant individuals. A similarly skewed distribution is observed in standing trees in a native population of P. radiata. The observed change in distribution cannot be attributed to ontological effects on resistance to pitch canker, because tree age accounts for almost none of the variation in susceptibility. We hypothesize that the change in distribution of susceptibility phenotypes is due primarily to infections and infection attempts by the microbiota resident in the forest.     

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

Fusarium 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.