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.     

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.     

Association of Sphaeropsis sapinea with insect‐damaged red pine shoots and cones

The association of the shoot blight and canker pathogen Sphaeropsis sapinea with red pine (Pinus resinosa) shoots and cones damaged by insects (especially Dioryctria sp.) was investigated. Samples from a single plantation approximately 35 years old, in Sauk Co., Wisconsin and also from three plantations, between approximately 40 and 50 years old, located in an area of pine shoot moth activity in the preceding year in Adams Co., Wisconsin were visually examined. Samples were arbitrarily collected from trees felled in the first plantation in May. Pycnidia of S. sapinea and insect damage were observed on 56 of 91 (62%) of closed cones and 17 of 165 (7%) of previous year's shoots. In the absence of insect damage, pycnidia of the pathogen were identified only on eight of 91 (9%) closed cones and never on previous year's shoots. In each of the other three plantations, 10 trees were located at intervals along transects in mid‐June; one branch from the lower half of the crown per tree was pruned off, and both current and previous year's shoots were examined. Insect damage and S. sapinea pycnidia were too rare on current year's shoots to draw any conclusions. Insect damage occurred on 20–40% of over 2000 previous year's shoots that were examined, but pycnidia of the pathogen were identified on only about 5%. Although infrequent, S. sapinea was identified in association with insect‐damaged previous year's shoots from these three plantations three times more frequently than those without insect damage. Random amplified polymorphic DNA (RAPD) markers from eight randomly selected isolates were consistent with the A group of S. sapinea, which can be aggressive on red pine. This ability to exploit insect‐damaged shoots may facilitate long‐term persistence of S. sapinea at low disease incidence and severity. The potential role of insect wounds as infection courts and insects as vectors of this important pathogen of pines deserves further study.     

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.     

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.     

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.     

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.     

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.     

Biocontrol of decay in seasoning utility poles. I. Growth rate and colonizing ability of bluestain and decay fungi in vivo and in vitro

Seasoning (air drying) of utility poles for 6–12 months is essential before preservative treatment can be achieved. However, during seasoning, pine sapwood is often colonized by decay fungi, thereby compromising the performance and service life of the poles. This study investigated the potential of bluestain fungi to act as short‐term biocontrol agents against decay during seasoning. An important attribute for biocontrol is rapid growth, so growth rates of common bluestain (Ceratocystis coerulescens, Ophiostoma minus, Ophiostoma piceae, Ophiostoma piliferum, Sphaeropsis sapinea) and decay fungi (Heterobasidion annosum, Phlebiopsis gigantea, Stereum sanguinolentum) were compared on agar medium and pine in logs at various temperatures. On agar, the growth temperature optimum of most bluestain fungi and all the decay fungi was ～25°C, with little growth at ≤5°C or above 32.5°C. Overall, the fastest growing were S. sapinea and O. minus. In logs, the most effective colonizers were S. sapinea and O. minus with pathogenic abilities that made them well fitted to colonize the sapwood of freshly felled pine. Within these species, certain isolates produced much larger lesions in phloem and the sapwood tangential plane than all the decay fungi. Notably, there was significant variation in colonizing ability between different isolates within a species, emphasizing the need for testing a range of isolates when selecting a potential biocontrol agent.     

The effects of long‐term exposure to simulated acid rain on the development of pine wilt disease caused by Bursaphelenchus xylophilus

To elucidate the synergetic effects of acid rain on the development of pine wilt disease, we measured the sap flow rate in the stems and the chlorophyll content in the needles of 10‐year‐old Japanese black pine trees, Pinus thunbergii and 12‐year‐old Japanese red pine trees, Pinus densiflora, after exposure to simulated acid rain (SAR, pH 3) or tap water (TW, pH 6.3) as a control. The heat pulse method was used for the estimation of the sap flow rate. No apparent difference was found in the sap flow rate between the trees exposed to SAR and TW, but the chlorophyll content of needles at the end of the treatment was significantly higher in the trees exposed to SAR than in those exposed to TW. When the pinewood nematode, Bursaphelenchus xylophilus, the causal agent of pine wilt disease, was inoculated onto the Japanese black pines that had been exposed to SAR repeatedly for 1 year, the period to death was shortened. Japanese red pines that had been exposed to SAR for 2 years, however, did not show any development of symptoms after the nematode inoculation, suggesting that acid rain only affects pine wilt disease slightly, if at all.     

Pre‐ and post‐inoculation water stress affects Sphaeropsis sapinea canker length in Pinus halepensis seedlings

In order to assess the influence of water stress on the development of Sphaeropsis sapinea cankers in Pinus halepensis, the stems of 4‐ to 5‐year‐old potted seedlings were artificially inoculated with the fungus before and after being kept at controlled water regimes from April 1997 to March 1998. In the pre‐water‐stress inoculation experiment, the canker length, measured 5 months after inoculation (September 1997), was greater in seedlings predisposed to extreme water deficit (midday needle water potential between ?4.5 and ?5.5 MPa). In the post‐water‐stress inoculation experiment, the fungus was inoculated in April 1998, after irrigation had enabled the seedlings to resume normal needle water potential. In this case also, at 5 months after inoculation, longer cankers were visible in seedlings that had been subjected to extreme water deficit. These findings suggest that the occurrence of marked water stress, although apparently tolerated by Aleppo pine, can enhance the development of S. sapinea cankers in this species, regardless of whether the stress occurs before or after infection by the fungus.     

Blue‐stain infections in roots,stems and branches of declining Pinus sylvestris trees in a dry inner alpine valley in Switzerland

Increased mortality rates in Scots pine (Pinus sylvestris) forests have recently been observed in the inner alpine Swiss Rhone valley. Drought, in combination with stand competition, mistletoe infections as well as nematode and insect infestations, appears to be the main factor for the decline. In focus of this study was the occurrence and role of fungal pathogens in the decline dynamics. Branches, stems and roots of 208 trees in five different crown transparency classes were collected and inspected for blue stain and fungal infections. Neither Armillaria species nor Heterobasidon annosum s. str. were detected, but blue stain was commonly observed. Visible blue stain increased with increasing crown transparency. Among the recently dead trees, 80% showed visible blue stain in the branches, 90% in the roots and 100% in the stems. In the crown transparency classes 2 and 3 (25–60% crown transparency), five of the 103 trees showed visible blue stain in the roots, one of 130 trees in the stem but none in the branches. Blue‐stain fungi were isolated from all parts of the trees and from all crown transparency classes. Overall incidence of blue stain was highest in the roots and lowest in the branches. In class 2, roots of 60% of the trees were visibly blue‐stained or developed blue stain in culture, but stems of only 24% and branches of 14% of the trees. In the roots Leptographium species, mostly L. serpens, dominated. From stems and branches, mainly Ophiostoma species were isolated. The positive relationship between the incidence of blue stain and crown transparency, in combination with the high infection levels of roots of fairly vigorous Scots pines, indicates the pathogenic potential of the blue‐stain fungi. Hence, these fungi together with their insect vectors may well act as an important contributing factor involved in pine decline.     

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.     

Mating type ratios and pathogenicity in Diplodia shoot blight fungi populations: Comparative analysis

Diplodia sapinea and Diplodia scrobiculata are opportunistic pathogens of Pinus species. Several studies about taxonomy, impact and epidemiology of these fungi have been conducted in previous years, which have provided useful information and have raised new issues. These diseases produce a considerable impact on plantations resulting in significant economic losses. The main aims of this study are to increase the knowledge of the potential of genetic exchange and the relative aggressiveness of these organisms that can persist in healthy tissues of asymptomatic trees. A collection of 250 isolates among which are 149 strains collected from Pinus radiata plantations in Basque Country (Spain) and 101 strains from different countries was included in this work. Mating type ratios were analysed and compared using the structure of the MAT locus (MAT1‐1‐1 and MAT1‐2‐1). Inoculations of Pinus radiata seedlings were performed in a biosafety greenhouse (P2) to confirm pathogenicity of isolates and compare their aggressiveness. The frequency of occurrence of both idiomorphs of D. sapinea in Basque Country isolates was close to 1:1, however, for collection of isolates of this fungus from around the world, the ratio was 1:2. Furthermore, the spatial distribution of the two mating types in the Basque Country was random. Despite no detection of a sexual state, these results could suggest sexual reproduction behaviour. The pathogenicity of all strains in the collection was confirmed. Although aggressiveness (in terms of lesion lengths resulting from inoculation) varied greatly, no statistically significant effects of MAT type or pathogen species were detected.     

A comparison of needle retention on Japanese black pine and Japanese red pine

Needle retention (number of needle sets), needle density, height increment and radial increment were surveyed on seven Japanese black pines (Pinus thunbergii Parl.) and seven Japanese red pines (Pinus densiflora Sieb. et Zucc.) growing on the same site. Number of needle sets on branches with respect to whorl position was estimated visually. In 1999, maximum summer needle retention was observed on the fifth and sixth whorl from the top, with values of 3.4 needle sets for the black pines and 2.4 needle sets for the red pines, respectively. The needle trace method (NTM) was used to determine needle retention and needle density along the main stems retrospectively for the years 1968–1998. The long-term mean summer needle retention along the main stem was 3.7 needle sets for the black pines and 2.2 needle sets for the red pines. In both pine species, the number of needle sets varied from year to year. However, the long-term budget between newly born and annually shed needle sets was in equilibrium. The long-term average of needle density per cm of stem shoot was 9.4 needle pairs for the black pines and 7.4 needle pairs for the red pines. The results showed clear intra-specific and inter-specific similarities in needle retention, height increment and radial increment trends. The values for number of needle sets, height increment and radial increment positively increased with favorable growing conditions, whereas the value of needle density had an opposite tendency.     

PCR primers for identification of Sirococcus conigenus and S. tsugae,and detection of S. conigenus from symptomatic and asymptomatic red pine shoots

Regions of diversity in the internal transcribed spacer (ITS) sequences of Sirococcus species were exploited to design primer pairs used in a PCR-based method for the identification of the conifer shoot blight pathogen Sirococcus conigenus and the closely related fungus Sirococcus tsugae. The specificity of each primer pair for the respective fungus, detection limits and utility for detection from host material were confirmed. The S. conigenus primers were then used to detect this pathogen in tissues of symptomatic or apparently healthy red pine shoots collected at six locations in Wisconsin and Michigan and results compared with those obtained using a cultural assay. For needles, bark and wood of symptomatic shoots, the mean frequencies of detection of S. conigenus using the PCR-based methods were consistent (≥7.5 out of 10) and always greater than for the cultural assay. Detection from symptomatic shoots using the cultural assay was more frequent from needles than from bark or wood. Both the PCR-based method and the cultural assay detected S. conigenus in similar frequencies from asymptomatic shoots, although less frequently than from symptomatic shoots. The efficiency of the PCR-based method and its utility for direct testing of host material should make it particularly useful in areas where multiple shoot blight pathogens are found.     

Development of Pinus sylvestris Main Stems Following Three Different Precommercial Thinning Methods in a Mixed Stand

Reducing competition by point cleaning may be an attractive alternative to conventional precommercial thinning in forestry. In this study, survival and development of main stems of Scots pine (Pinus sylvestris L.) following point cleaning were examined in a mixed stand of mainly Scots pine and birch (Betula pendula Roth and Betula pubescens Ehrh.) in northern Sweden. Treatments studied were: no precommercial thinning, conventional precommercial thinning, point cleaning of all secondary stems, and point cleaning of only the secondary stems that were higher than the main stem, within a radius of 0.8 m from the main stem, respectively. A randomised complete block design with three replicates was used. Measurements taken 13 growing seasons after these treatments showed that conventional precommercial thinning was the best treatment for retaining Scots pines as main stems. However, conventional precommercial thinning resulted in the poorest timber quality of the remaining Scots pines in terms of stem taper, thickest branch diameter and relative live crown height. In conclusion, point cleaning has to be followed by a complementary cleaning operation if the objective is to retain Scots pines as main stems in such mixed stands.     

Browsing and damage inflicted by moose in young Scots pine stands subjected to high-stump precommercial thinning

In Fennoscandia, young stands of Scots pine (Pinus sylvestris L.) are intensively used by moose (Alces alces L.) during winter. We studied whether forage amounts on high-cut pines in high-stump commercial thinning influenced browsing intensity and damage incidence on retained (i.e. uncut) pine stems. High-cut pines were browsed, but to a lesser extent than retained pines. At a scale corresponding to individual feeding sites (≈40 m²), browsing intensity on retained pines was not influenced by the amount of forage on high-cut pines but was positively related to moose pellet group counts. The incidence of lower-height damage (stem breakage and bark stripping) was positively related to the amount of forage on high-cut pines, whereas higher damage (leader shoot browsing) was not. Overall browsing damage incidence on retained pines was positively related to the density of deciduous trees and negatively related to the amount of forage on retained pines. Our results suggest that although high-stump thinning supplies additional food resources for moose, larger amounts of forage on high-cut pines may increase the risk for bark stripping and stem breakage on retained trees. Further research is needed at larger spatial scales to assess the feasibility of high-stump thinning as a damage mitigation measure.