Risk from Sirococcus conigenus to understory red pine seedlings in northern Wisconsin

Interest in development of multicohort stands of red pine (Pinus resinosa) in the Great Lakes region of North America prompted an investigation of the potential impact of the shoot blight pathogen Sirococcus conigenus (syn. S. strobilinus) on understory red pine seedlings. In May 2002 and 2003 healthy, 1‐year‐old red pine seedlings were planted in the understory of a maturing red pine plantation in northern Wisconsin in an area with a history of presence of this pathogen. Occurrence of shoot blight symptoms was recorded periodically during the summer, and in each year seedlings were harvested in fall and examined for signs of shoot blight pathogens. By fall 2002 and 2003, respectively, shoot blight incidence was 89% and 98% and most seedlings were dying. Pycnidia with conidia of S. conigenus were present on almost all of the symptomatic seedlings. The conifer shoot blight and canker pathogen Diplodia pinea (syn. Sphaeropsis sapinea) was also detected, though less frequently. Pycnidia of S. conigenus tended to be found more frequently on symptomatic current year's shoots than symptomatic previous year's shoots; the opposite was true for pycnidia of D. pinea. Risk from S. conigenus to understory red pine seedlings should be considered in any plans for development of multicohort red pine stands in areas where the pathogen is present.     

Site-related influences on cone-borne inoculum and asymptomatic persistence of Diplodia shoot blight fungi on or in mature red pines

The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata sporulate abundantly on cones of many pine hosts. Variation in incidence and abundance of potential inoculum from cones and frequency of asymptomatic persistence on or in shoots was examined for mature red pines in sites differing in dominant presettlement vegetation and soil type in Bayfield and Douglas counties in northern Wisconsin. Collections were made in each county from 6 plantations, 3 each in areas historically vegetated with jack pine and soils mapped as sands and three in areas historically vegetated with red pine with soils mapped as loamy sands. At each site, 5 cones were collected from each of 5 red pines and 10 shoots were collected from up to 5 red pines. Conidia from cones were quantified with a water wash and filtration technique. Diplodia species were cultured from surface-disinfested asymptomatic shoots. A species-specific PCR assay was used to identify the Diplodia species from cones and shoots. Although cones and asymptomatic shoots from each county yielded D. pinea and D. scrobiculata, D. pinea was detected more frequently. More conidia were obtained from cones from Douglas Co., where there is a history of severe shoot blight damage, than cones from Bayfield Co. In Douglas Co., more conidia were obtained from cones from plantations in areas of more sandy soil and presettlement jack pine dominance than cones from plantations in areas of less sandy soil and presettlement red pine dominance. The numbers of conidia and frequencies of cultural detection of Diplodia species from asymptomatic shoots at a site were positively correlated. These results provide evidence for site-related influences on abundance of pathogen inoculum and asymptomatic persistence on or in red pine crowns that may contribute to differences in frequency and severity of damage from Diplodia shoot blight.     

An effective medium for isolating Sphaeropsis sapinea from asymptomatic pines

The pathogen Sphaeropsis sapinea can persist in stems and branches of asymptomatic pines and can later induce disease when triggered by host stress. Several experiments were conducted to test if:(i) medium amended with tannic acid (TA) can increase the frequency of cultural detection of this shoot blight and canker pathogen from asymptomatic red pine (Pinus resinosa) stems, and (ii) S. sapinea can persist in asymptomatic red pine in the field following artificial inoculation. TA (0.5% w/v) in 2% (w/v) water agar proved to be the best medium for isolation of S. sapinea among a larger number of tested media. The addition of TA had little or no effect on the growth of two group A and two group B isolates of S. sapinea. However, when TA was added, 11 other fast‐growing fungal isolates from stems/branches of red or jack pines (P. banksiana) were inhibited and grew more slowly (p < 0.05) than both S. sapinea groups. The TA‐amended medium improved cultural detection of S. sapinea from 2‐year‐old, asymptomatic red pine nursery seedlings compared with two other methods used for the cultural detection of S. sapinea (32%vs. 8.5% and 18% recovery; p < 0.001 and p = 0.031, respectively). A field test using the TA‐amended media established that S. sapinea can persist asymptomatically in red pine trees for at least 1 year. This medium significantly reduces the frequency of false‐negatives from asymptomatic field material.     

Surveys for asymptomatic persistence of Sphaeropsis sapinea on or in stems of red pine seedlings from seven Great Lakes region nurseries

The fungus Sphaeropsis sapinea persists on or in stems of asymptomatic red pine (Pinus resinosa) nursery seedlings, and proliferates to cause collar rot and mortality after planting. In the spring of 2002, seven nurseries were surveyed to determine the potential range in frequency of asymptomatic persistence: three operated by Wisconsin Department of Natural Resources (DNR), two by Minnesota DNR, one by Michigan DNR, and one by USDA Forest Service (in Michigan). At each nursery five groups of 20 asymptomatic red pine seedlings were collected near an inoculum source (red pine windbreak), if present, and five groups of 20 asymptomatic seedlings were collected away from such a source (1400 seedlings total). A segment of the lower stem/root collar from each seedling was surface disinfested and incubated on tannic acid agar. Transfers were made from resulting colonies and the pathogen identified from pycnidia and conidia produced in culture. The pathogen was identified from asymptomatic seedlings collected in all Wisconsin and Minnesota nurseries, but was never detected from seedlings from the Michigan DNR or USDA Forest Service nurseries. Frequencies of detection were greater (as high as 88%) from asymptomatic seedlings near red pine windbreaks including diseased trees than from seedlings distant from such windbreaks. A subset of isolates from asymptomatic seedlings was characterized using inter‐simple sequence repeat–polymerase chain reaction analysis. Most isolates were the A group of S. sapinea, but B group isolates (recently named Diplodia scrobiculata) were also obtained from one nursery. One Minnesota nursery was more extensively sampled in 2003, with 17–44 groups of five asymptomatic red pine seedlings collected in four separate fields (525 seedlings total). The mean frequency of detection of the pathogen in these four fields ranged from 40 to 71%. Persistence of S. sapinea on or in asymptomatic seedlings continues to be problematic, not only because of the potential for subsequent seedling mortality, but also as a means by which a pathogen may be widely distributed.     

Persistence of some pine pathogens in coarse woody debris and cones in a Pinus pinea forest

The persistence of Sphaeropsis sapinea, Leptographium serpens and Heterobasidion annosum s.s. in artificially inoculated pine branch pieces (S. sapinea and L. serpens) and wood blocks (L. serpens and H. annosum s.s.) was investigated in order to discuss the alternative of leaving coarse woody debris in stands of Italian stone pine (Pinus pinea). Also, natural colonization by S. sapinea of pine cones of different ages was assessed. Methods used for inoculating branch pieces and wood blocks were highly effective for all fungi. Type of a forest stand in which branch pieces and wood blocks have been incubated did not affect the persistence of the pathogens in the inoculated samples. For branch pieces, the success of re-isolation of L. serpens dropped as the sample incubation time increased, while S. sapinea was always successfully (100%) re-isolated (even 12 months after the inoculation). L. serpens and H. annosum s.s. were re-isolated from most of the buried wood blocks (from more than 95% samples) up to 3 months following the inoculation. Of the observed P. pinea cones (in most cases, more than 2 years old), 74% were naturally infected byS. sapinea. All three investigated pathogens were able to survive in dead plant tissues for long periods of time (at least for several (3–12) months). The persistence of these pine-pathogenic species in dead plant material questions the feasibility of leaving coarse woody debris in managed Italian stone pine forests meant for landscape conservation and leisure activities.     

Longevity of inoculum production by Diplodia pinea on red pine cones

In summer of 2004, pycnidia of Diplodia pinea were observed on cones of Pinus resinosa that had matured and opened during previous years, but had been retained in canopies of trees at a mature red pine plantation in southern Wisconsin. Surveys during the winter and early summer of three consecutive years (2005–2007) to determine incidence and abundance of D. pinea conidia on cones of different ages in this stand. Cones from each age class consistently bore pycnidia with conidia of D. pinea. Although cones collected in June of the year after their maturation tended to yield more D. pinea conidia than older cones, large numbers of conidia were obtained from cones even 3 years after maturation. Perennial availability of inoculum due to persistence of D. pinea on cones of several ages in the overstory or in adjacent stands should be considered when regenerating red pine in areas where this pathogen is known to be present.     

Infection approach of Diplodia sapinea,the causal agent of tip blight of Pinus tabulaeformis in China

To clarify the infection approach of Diplodia sapinea, a pathogen that causes tip blight of Pinus tabulaeformis, the infection process of the pathogen in needles was observed using scanning electron microscopy (SEM). In addition, the disease incidence on branches damaged by Aphrophora flavipes (Hemiptera: Cercopidae) and Dioryctria splendidella (Lepidoptera: Pyralidae) in the forest was also investigated. Then, branches and needles of P. tabulaeformis were inoculated using the D. sapinea spore suspension under indoor and field conditions. The results showed that the damage caused by A. flavipes could aggravate the occurrence of tip blight of P. tabulaeformis to some extent. Moreover, the pathogen could also penetrate 1‐, 2‐ and 3‐year‐old pine needles through stomata in the field. The pathogen infected the 1‐year‐old branches first and then gradually spread to 2‐ and 3‐year‐old branches.     

Genotypic diversity and distribution of Sphaeropsis sapinea within Pinus radiata trees from northern Spain

Sphaeropsis sapinea is an important latent pathogen of Pinus spp., outbreaks of which have a considerable impact on plantations. This study considers the population diversity and distribution of S. sapinea in northern Spain at different spatial scales from single plantations to a wide area covered by Pinus radiata trees. Estimation of genotypic diversity is an important component of the analysis of the genetic structure of plant pathogen populations. Ten simple sequence repeat (SSR) markers were used, together with vegetative compatibility tests, to study the genetic diversity among S. sapinea isolates. Polymorphism analysis at SSR loci is a simple and direct approach for estimating the genetic diversity of S. sapinea isolates. From a total of 86 isolates collected from four different areas, 14 microsatellite haplotypes and 13 vegetative compatibility groups (VCGs) were identified. The percentage of maximum genotypic diversity, based on Stoddart and Taylor's index, for microsatellites of the northern Spain population ranged from 14.6% to 38.1% and from 8.0% to 29.4% for VCGs. Analysis of these markers and vegetative compatibility groups confirmed that S. sapinea reproduces mainly asexually due to its reduced genotypic diversity in spatially close populations. Isolates of S. sapinea from northern Spain populations were predominantly monomorphic at the tested SSR loci. Vegetative compatibility groups also indicate a low level of genetic variability in these samples, which appear to be clonal.     

Detection of Diplodia pinea in asymptomatic pine shoots and its relation to the Normalized Insolation index

Diplodia pinea (syn. Sphaeropsis sapinea), a common pathogenic fungus, causes considerable damage in Italy, particularly to pine stands in which trees are subjected to environmental stress. The occurrence of D. pinea in symptomless Pinus nigra shoots was investigated and related to the amount of radiation received by the trees growing on a site in a year, expressed as the Normalized Insolation index (NIi). Twenty‐seven pines were selected from nine locations in Trentino (northern Italy). For each pine the incidence of the fungus in apparently healthy shoots was determined by both culturing on an agar medium and application of real‐time PCR. The incidence of D. pinea determined by culturing samples taken from asymptomatic trees was 59% (16 of 27 trees), compared with 85% found using real‐time PCR (23 of 27 trees). Detection of the pathogen in healthy pine tissue was positively correlated (p < 0.05) with the NIi values, using both detection methods.     

Sphaeropsis shoot blight and altered nutrition in red pine plantations treated with paper mill waste sludge

During 1993 progressive, severe shoot blight and canker disease in crowns of mature, merchantable red pines (Pinus resinosa) in central Wisconsin was noted in plantations in which paper mill waste sludge previously had been applied. For eight treated plantations and six non‐treated plantations, incidence of shoot blight attributed to the pathogenic fungus Sphaeropsis sapinea (syn. Diplodia pinea) was quantified during 1993 or 1994. Foliage and soil samples also were collected for analyses. Sphaeropsis shoot blight was more frequent in treated plantations than in the non‐treated stands (means of 81% of trees and 10.2% of shoots compared with 7% of trees and 0.1% of shoots, respectively). Consistent with other reports of damage caused by some diseases of conifers in situations of altered host nutrition, mean foliar N concentrations were higher in treated plantations (1.61%) than non‐treated plantations (1.31%) (p < 0.001). Mean foliar Zn, Mn, Cu, and Al concentrations were lower in treated plantations than non‐treated plantations, and mean soil P, Ca, and Mg concentrations were higher in treated plantations than non‐treated plantations.     

全球松树枯梢病发生状况与防治策略

松树枯梢病是一种世界性病害。该病寄主范围广,可侵染8个属约60种（含变种）针叶树。对全球松树枯梢病为害较严重国家（新西兰、南非、美国和中国等）的发生状况以及近年有关该病发生发展的流行诱因和抗性研究等进行了概述,并提出以树种生态适应性、提高寄主生长势为主以及控制病菌增量的防治策略。     

Weevil feeding on Scots pine affects germination of Endocronartium pini

The aim of this study was to assess the effect and importance of the feeding of the pine top weevil (Pissodes piniphilus) on the germination of Endocronartium pini (syn. Peridermium pini) in Scots pine (Pinus sylvestris), and thus to establish the possibility of the E. pini infection via insect woundings. Germination tests were therefore carried out on current and previous year needle and phloem extracts. Elucidation of the importance of pathogen infection for the insect's feeding preference was also required; for this purpose feeding preference tests were carried out with healthy and infected pine branches using the pine top weevil as a test insect. Weevil feeding increased the germination of E. pini aeciospores on pine extracts. Germination on previous year annual-shoot extracts was lower than that on current year annual-shoot extracts. The advance of the growing season increased this trend, but weevil feeding increased germination on extracts from older annual shoots to the levels found on extracts from current annual shoots. Spores germinated equally well on needle extracts and on phloem extracts. The weevils ate more often on infected branches than on healthy branches. E. pini infections may occur via woundings on branches and weevil feeding may facilitate this.     

Diplodia shoot blight and asymptomatic persistence of Diplodia pinea on or in stems of jack pine nursery seedlings

Diplodia pinea (syn. Sphaeropsis sapinea) is known as a major cause of damage to red pine (Pinus resinosa) seedlings in nurseries. The fungus can also be a latent pathogen of red pine seedlings, persisting in the absence of gross symptoms and later proliferating under conditions that induce host stress. In the fall of 2004, three nurseries in Wisconsin were surveyed to determine the potential for the occurrence of Diplodia shoot blight on jack pine (Pinus banksiana) seedlings and the persistence of D. pinea on or in asymptomatic seedlings of this species. Incidence of shoot blight was quantified in five 1 m long segments of an interior row in each of two survey areas in each nursery. The pathogen was identified on symptomatic seedlings collected in these areas on the basis of presence of characteristic pycnidia and conidia. Five groups of 20 asymptomatic seedlings were also collected in each of the two survey areas in each nursery. A segment of the lower stem/root collar from each of these asymptomatic seedlings was surface‐disinfested and culturally assayed using tannic acid agar. The mean incidence of shoot blight (as high as 9%) and mean frequency of cultural detection from asymptomatic seedlings (as high as 20%) were greatest in proximity to red pine windbreaks which are a source of inoculum. Only D. pinea was confirmed from subsets of symptomatic and asymptomatic seedlings which were tested using mt SSU rDNA polymerase chain reaction (PCR) primers that allow differentiation of D. pinea from D. scrobiculata and other fungi in the genus Botryosphaeria and related anamorphic fungi. Jack pine seedlings inoculated with D. pinea isolates obtained from asymptomatic nursery seedlings developed shoot blight symptoms in greenhouse trials. Thus, the ability of D. pinea to damage jack pine seedlings in nurseries has been documented and the potential for virulent strains of this latent pathogen to be distributed on asymptomatic jack pine seedlings from nurseries has been confirmed.     

New and unexpected host associations for Diplodia sapinea in the Western Balkans

Diplodia sapinea is an important pathogen of pine trees in plantations and urban areas in many parts of the world. This pathogen has recently also been isolated from diseased Cedrus atlantica, C. deodara and Picea omorika planted as ornamentals across the Western Balkans. The aim of this study was to consider the host range of D. sapinea in Serbia and Montenegro. Diplodia sapinea was identified from a broader collection of Botryosphaeriaceae from the Western Balkans region, based on the DNA sequence data for the internal transcribed spacer (ITS) rDNA and the translation elongation factor 1α (TEF 1‐ α). The D. sapinea isolates were obtained from sixteen tree species in the genera Abies, Cedrus, Chamaecyparis, Juniperus, Picea, Pinus, Pseudotsuga and Fagus. Four species represented new hosts in the Balkans, and this is the first report of D. sapinea from F. sylvatica anywhere in the world. Pathogenicity tests were conducted on the tree hosts from which D. sapinea was isolated, as well as on P. abies, Thuja occidentalis, Prunus laurocerasus, Eucalyptus grandis and P. patula. Inoculations were made on seedlings in the field, in the greenhouse or on freshly detached branches. Inoculations on P. pungens, P. omorika, P. abies, P. menziesii, A. concolor, P. nigra and P. sylvestris resulted in death of the seedlings 5–16 weeks after inoculation. Diplodia sapinea produced lesions on J. horizontalis and P. patula seedlings and F. sylvatica cut branches. Reciprocal inoculations showed that D. sapinea is not a pine‐specific pathogen, causing disease on tree species, including those from which it had not been isolated. Not surprisingly, the pathogen was most aggressive on some species of Pinaceae.     

Variation among New Zealand isolates of Sphaeropsis sapinea

Sphaeropsis sapinea is a pathogen of many coniferous species and causes significant losses to the plantation forestry industries of many countries. New Zealand isolates of S. sapinea were examined for colony and morphological characteristics, and pathogenicity to Pinus radiata seedlings. Considerable variation was observed in colony growth form and colour, and in the growth rates on four agar media. The conidial dimensions, carbon and nitrogen utilization, and pathogenicity also varied among the isolates. The variation observed was such that the isolates could not be placed within previously described distinct morphotypes and the present results concur with previous studies cautioning the use of morphological criteria alone to partition the species. These results contrast markedly with previous reports and seemingly indicate a change in the New Zealand S. sapinea population. This difference may reflect the re‐introduction or spread of additional isolates or a shift in the structure of the resident population.     

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash in northern Sweden

Survival and vitality of Gremmeniella abietina on Pinus sylvestris slash was studied in northern Sweden during 2003 and 2004. Once a month between September 2003 and April 2004, two to three trees were cut down and debranched. Shoots with pycnidia were sampled at the felling date and then at every consecutive month. The percentage of germinated conidia from each shoot was calculated after 24, 48 and 72 h incubation. The vitality of G. abietina pycnidia in the slash remained high the whole period. Intact pycnidia were found on slash several months after the time of conidial sporulation, which indicates that new pycnidia may be produced on dead pine branches. Sampling of shoots from slash on 13‐ to 18‐month‐old clear‐cuts showed conidial germination capacity as high as in pycnidia collected in fresh slash. Due to survival of G. abietina in slash it is recommended to postpone planting of P. sylvestris seedlings in northern boreal areas to the third vegetation period after sanitary clear‐cuts.     

Pathogenicity of A and B morphotypes of Sphaeropsis sapinea confirmed on American larch (tamarack) and European larch

Fungi identified as Sphaeropsis sapinea (or synonyms) have been reported from Larix. Lack of pathogenicity tests, confusion about the identity of isolates mentioned in previous literature, and existence of distinct populations (A and B morphotypes) of the pathogen indicated the need to evaluate the ability of S. sapinea to cause disease of larch. Elongating snoot tips of Larix decidua, Larix laricina, and known hosts of the pathogen, Pinus banksiana and Pinus resinosa, were inoculated with water-agar plugs colonized with an A or a B isolate, or sterile plugs (controls). Each of five replications included five seedlings for each treatment-species combination. After 6 weeks, no symptoms had developed on control seedlings, but two-way analyses of variance revealed significant effects of isolate morpnotype and host on both incidence and severity of disease (values of p ≤ 0.01). The A isolate killed almost all shoot tips, but the B isolate killed from no P. resinosa shoots to 56% of L. laricina shoots. The average length of shoot killed by the A isolate was also greater than that killed by the B isolate. Response to the B isolate again varied among species, with greater average lengths of shoot killed on the larches (compared with the pines). Both A and B morphotypes of 5. sapinea should be considered among the fungi encountered on L. decidua and L. laricina.     

Frost resistance and frost damage in pinus sylvestris seedlings during shoot elongation

No differences were found in the frost resistance of the different‐aged (2–7 weeks) germlings of Scots pine. The critical temperature was ‐6°C to —3°C. A temperature of ‐4°C killed half of the germlings and produced damage in one fifth of the surviving seedlings; the needles turned brown and growth ceased. The frost resistance of the previous year's shoots of two‐year‐old seedlings, measured by the impedance method, decreased during the shoot elongation period from — 10°C to — 4°C. Damage to the current shoots during shoot elongation appeared as deformation and discolouration of the needles and abnormal development or death of the apical bud. The results indicate that frost is not the cause of growth disturbances observed in Finnish nurseries.     

The aggressiveness of Sphaeropsis sapinea on Austrian pine varies with isolate group and site of infection

The aggressiveness of the A and B isolate groups (morphotypes) of Sphaeropsis sapinea on shoot tips and lower stems of Austrian pine (Pinus nigra) was compared. The distinct differences in aggressiveness between the two S. sapinea groups, previously reported for other conifers, are confirmed for Austrian pine. However, the relative aggressiveness of the B group isolate varied by site of infection. Although the B group isolate of S. sapinea was not aggressive on shoot tips of Austrian pine, it was aggressive on stems of this host.