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.     

Evidence for the occurrence of induced resistance to pitch canker,caused by Gibberella circinata (anamorph Fusarium circinatum), in populations of Pinus radiata

Pitch canker, caused by Gibberella circinata, was discovered in California in 1986. Although initially quite damaging to Monterey pines (Pinus radiata), the severity of pitch canker has moderated in areas where the disease was first observed and some trees appear to have recovered completely. The absence of symptoms on trees that were once severely affected implies they have become more resistant to the disease. Experimental work has shown that P. radiata can manifest systemic induced resistance (SIR) in response to infection by the pitch canker pathogen and observations of disease remission may indicate that SIR is operative under natural conditions as well. As a test of this hypothesis, the susceptibility of trees in remission was assessed by inoculating them with G. circinata and recording the extent of lesion development. In addition, randomly selected trees in areas that differed in residence time of pitch canker were inoculated to determine if trees with a longer period of exposure to the pathogen were more resistant to the disease. The results of these tests showed that 89% of trees observed to be in remission sustained very limited lesion development, consistent with resistance to pitch canker. Furthermore, trees in areas where pitch canker was well established tended to be more resistant than trees in areas where the disease was of more recent occurrence. In sum, these findings support the view that SIR occurs in P. radiata and is contributing to a moderation of the impact of pitch canker under natural conditions.     

Pitch canker ratings of longleaf pine clones correlate with Fusarium circinatum infestation of seeds and seedling mortality in containers

The incidence and severity of pitch canker was rated among 16 clones of mature longleaf pine (Pinus palustris) in a seed orchard and cones were collected from five of those clones across the range of pitch canker disease ratings. Seed were extracted and commercially processed by clone and the percentages contaminated with Fusarium circinatum determined. Seed from each clone were sown either without treatment or with one of three fungicide treatments in a soil‐less mix at a commercial container nursery to evaluate the effects of F. circinatum (syn. F. subglutinans) on seed and seedling survival. The percent of seed with F. circinatum correlated with the pitch canker ratings in the orchard for the year of collection (r = 0.88, p = 0.05) and, when sown without fungicide treatment, with the number of seedlings produced (r = ?0.94, p = 0.01) and with seedling mortality after germination (r = 0.92, p = 0.02). The same orchard clones were more symptomatic of pitch canker through three annual surveys. Fusarium circinatum was isolated from a higher percent of seed from more symptomatic clones and a lower percent of their seed produced plantable seedlings. We propose that removing such clones from seed collections should reduce seedling mortality at the nursery.     

High genetic diversity of Fusarium circinatum associated with the first outbreak of pitch canker on Pinus patula in South Africa

The disease known as pitch canker results from infection of Pinus species by the fungus Fusarium circinatum. This fungus also causes a serious root disease of Pinus seedlings and cuttings in forestry nurseries. Pinus radiata and P. patula are especially susceptible to the pathogen, but there are no records of pitch canker on P. patula in established plantations. To date, only planting material of this tree species in nurseries or in plantations at the time of establishment have been infected by F. circinatum. Symptoms of pitch canker have recently emerged in an established P. patula plantation in South Africa and this study sought to determine whether the symptoms were caused by F. circinatum. Isolates from cankers were identified as F. circinatum using morphology and DNA-based diagnostic markers. Microsatellite markers were then used to determine the genetic diversity of a collection of 52 isolates. The entire population included 17 genotypes representing 30 alleles, with a greater number of genotypes collected from younger (three- to six-year-old) than older (12- to 19-year-old) trees. Both mating types of F. circinatum were present, but no evidence of sexual recombination was inferred from population genetic analyses. This is the first record globally of pitch canker on P. patula trees in managed plantations. It is of significant concern to South Africa, where P. patula is the most important Pinus species utilised for plantation forestry.     

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.     

Differential responses of Central American and Mexican pine species and Pinus radiata to infection by the pitch canker fungus

Seedlings from a wide array of 23 species, varieties,and geographic races were screened for resistance topitch canker using artificial inoculation in agreenhouse. Seed to represent these taxa weregenerally collected in natural stands. In addition,seedlings from 79 families of P. radiata fromcommercial populations from Chile and New Zealand werescreened in a separate experiment. There was littlevariation in resistance among the commercial P.radiata families, with over 98% mortality. Similarresults were obtained with native P. radiatapopulations. All close relatives of P. radiata(sub-section Patula) were very susceptible. However,a number of closed-cone pine species (specifically,from sub-section Oocarpa) were generally veryresistant, with survival approaching 100%. Inaddition, in P. oocarpa, P. jaliscana, and P. tecunumanii from low elevation provenances anextremely high frequency of trees suffered no apparentdamage from the pathogen (92%, 85% and 80%,respectively).     

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.     

AFLP analysis of Fusarium circinatum and relative species

AFLP fingerprinting analysis of Fusarium circinatum, Nirenberg and O’Donnell and relative species was carried out. Ten primer-pairs that could generate abundant polymorphism fragments were screened. A total of 298 nucleotide acid fragments were amplified with the primers from the template of the 17 strains of Fusarium spp., among which 283 fragments were polymorphic. Percentage of polymorphic loci produced by each pair of AFLP primer-pair was 94.97% in average and varied from 89.29% to 100%. All these data indicated that considerable genetic variation existed among F. circinatum and relative species at DNA level. Molecular genetic distances among Fusarium spp. were calculated, and the relationship among them was described quantitatively. Compared with biological species, the result of cluster analysis was basically similar to the phenotypic classification of species. Genetic diversity of E-AT/M-CAA AFLP fingerprinting of Fusarium spp. was analyzed, and specific and difference bands for each species and all Fusarium section Liseola tested were identified based on the E-AT/M-CAA AFLP fingerprinting.     

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.     

Relation between wilt rate and obstruction of water flow in stems of two families of loblolly pine affected by pitch canker

Movement of water forced through stem segments of two half-sib families of loblolly pine (Pinus taeda), infected by Fusarinum subglutinans, was monitored. Wilt rate of the two families was shown to be correlated with obstruction of water flow through stem segments of similar disease severity.     

Susceptibility of six pine species to Gremmeniella abietina isolates from Spain

Inoculation experiments were performed in order to evaluate the virulence of Gremmeniella abietina isolates from Spain on the main pine species planted in the Iberian Peninsula, as well as the influence of seedling age on this virulence. Two different experiments were carried out with four isolates of G. abietina from Spain. The greenhouse experiments consisted of seedling inoculations. One‐ and 2‐year‐old seedlings of the following five pine species were used: Pinus halepensis, Pinus pinea, Pinus pinaster, Pinus sylvestris and Pinus uncinata; also, 1‐year‐old seedlings of P. nigra were inoculated. The relative necrosis length (RNL) caused by the pathogen after 130 days was used as a response variable. The laboratory experiments were performed on 2‐ to 6‐year‐old internodes of the above pine species excluding P. uncinata. The necrosis length after 6 weeks of incubation was measured. The results have shown that all G. abietina isolates were pathogens on seedlings of these six pine species and seedlings of P. halepensis were consistently the most susceptible ones, although it is important to take into account that all the isolates used in the present work were isolated from P. halepensis, the only pine species in Spain where G. abietina has been recovered up to now. The susceptibility of the other pine species depended on the age of the seedlings.     

Decay and canker caused by Inonotus rickii spreading on more urban tree species

Inonotus rickii was detected for the first time causing cankers and decay in Acer negundo and Celtis australis in Italy. In a boxelder boulevard, declining trees showed sparse foliage, exudations and cracks in the bark; in some cases, chlamydospore masses were present. Five isolates were collected and compared by growth tests in vitro and electrophoretic analyses; three isolates from the same boulevard showed very similar physiological characters. The increasing importance of the pathogen in urban areas is underlined and discussed.     

Relative susceptibility of several European species of pine to fusiform rust

The relative susceptibility to fusiform rust of provenances of 6 European species of pine was tested at Athens, Georgia U.S.A. Needle and stem spots caused by rust were found on the seedlings of all the species and their provenances. Several maritime pine and Aleppo pine seedlings showed particular longitudinal cracks caused by the fungus mycelium in the stem; those of Austrian pine and of Swiss Mountain pine had a violet zone around the cotyledons due to the presence of the fungus mycelium in the needle tissues. If the percentage of galled seedlings is taken into consideration, the test results indicate that Aleppo pine, Austrian pine and Italian stone pine are more susceptible to infection than are maritime pine, Swiss Mountain pine or Scots pine.     

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.     

Needle blight of pine caused by two species of Dothistroma in Hungary

Dothistroma septosporum and D. pini cause the serious pine needle disease, Dothistroma needle blight (DNB). Of these, D. septosporum has a global distribution, while D. pini is known only from the USA, Russia, Ukraine and France. During a study of D. septosporum isolates from Pinus nigra in Hungary, microsatellite markers revealed the presence of a second Dothistroma species. The aim of this study was to identify the DNB pathogens occurring in Hungary using four different molecular techniques. These included sequencing of the rDNA ITS region, a species‐specific ITS‐RFLP, mating type primers and a diagnostic microsatellite marker, Doth_A. Results showed that both D. septosporum and D. pini occur in Hungary and that both DNB pathogens were present on the same trees and in some cases, the same needles. Mating types MAT1 and MAT2 of D. septosporum were shown to be present in Hungary, but only the MAT2 of D. pini was found. In addition, examination of needles with DNB symptoms from Russia revealed the presence of D. pini on Pinus pallasiana and P. nigra as well as on a new host, Pinus mugo. The molecular markers applied in this study were sufficiently robust to identify and differentiate between the two DNB pathogens, both in culture and directly from needles. They will consequently be useful to document the geographical range and to monitor the spread of D. septosporum and D. pini in future studies.     

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.     

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.     

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.