Red pine harvest debris as a potential source of inoculum of Diplodia shoot blight pathogens

A high incidence of Diplodia shoot blight (site means ranging 85-100%) was observed on recently planted red pine (Pinus resinosa) seedlings where mature red pine stands previously had been clearcut. An investigation of the potential of harvest debris as a source of inoculum of Diplodia pathogens then was conducted. Cones, bark, needles, stems from shoots bearing needles, and stems from shoots not bearing needles (both suspended above the soil and in soil contact) were collected from harvest debris left at sites where clearcutting occurred. Conidia were quantified, and their germination rate was assessed, and Diplodia species were identified using PCR. Conidia of Diplodia species were found at all study sites and conidia counts increased from samples collected from 6 to 18 months after harvest. Germinable conidia were obtained from debris collected 6 months to 5 years after harvest. Fewer conidia were obtained from debris collected at intervals of up to 4-5 years after harvest and the percentage of germinable conidia was lower after longer intervals following harvest. More conidia were obtained and a greater percentage germinated from debris collected above the soil than from debris in soil contact. The host substrate also influenced the number of conidia and the percentage that germinated. Planting red pine seedlings next to debris infested with Diplodia pathogens could provide a persistent source of inoculum. Results should prompt further consideration by land managers and researchers of the potential forest health risks, in addition to benefits, that may be associated with harvest debris.     

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.     

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.     

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.     

The pine pathogen Diplodia sapinea is associated with the death of large Douglas fir trees

Climate change poses severe pressures to European conifer forests. Using non-native tree species, such as Douglas fir (Pseudotsuga menziesii), is one proposed strategy to circumvent adverse effects for forest management. However, novel forest health risks can impair the cultivation of non-native trees. In 2022, we observed large Douglas fir trees (approximately 40–50 years old, diameter at breast height (dbh) 21–41 cm) that had recently died in spring or summer 2022 in three forest stands in Eastern Austria. Intensive resin flow, blue-staining of the sapwood and the absence of bark- and wood-boring insects indicated a fungal infection. Isolations from blue-stained sapwood of the dead trees consistently yielded cultures of the opportunistic pathogen Diplodia sapinea. In a greenhouse wound inoculation experiment, seven D. sapinea isolates obtained from Douglas fir caused phloem necrosis, blue-staining of sapwood and mortality and thus displayed pathogenicity towards seedlings of both Ps. menziesii and its common host, Scots pine (Pinus sylvestris). Diplodia sapinea produced significantly longer areas of blue-stain as well as higher and faster mortality in Ps. menziesii compared to P. sylvestris. We conclude that D. sapinea substantially contributed to the death of seven of the 13 examined large Douglas fir trees. While this fungus has been described as a pathogen of young Douglas fir trees before, this is the first report that it can potentially kill large individuals of this conifer species under drought conditions. Thus, our results indicate that D. sapinea could represent a severe threat to the cultivation of Ps. menziesii in European forestry.     

Growth responses of red pine seedlings to the chemical bioregulator,DCPTA

The chemical bioregulator, 2-(3, 4-dichlorophenoxy)-triethylamine (DCPTA), which has markedly stimulated photosynthesis, carbon allocation, and above- and below-ground growth of several agricultural crops, caused small growth effects on red pine (Pinus resinosa L.) germinants and one and two-year-old seedlings, mainly to foliage and stems. At low DCPTA concentrations, i.e. 10 ppm, growth tended to be promoted, whereas at 100 ppm growth appeared to be suppressed. Continued testing of the bioregulator's effect on growth and physiology of other tree species is recommended.     

Infection of Scots pine seedlings by Gremmeniella abietina during summer under different inoculum potential

The connection between natural conidia dispersal of Gremmeniella abietina (A‐type) and the disease incidence and severity in first and second year conifer seedlings of Pinus sylvestris was studied in central Finland. The seedlings where exposed to natural infection for a 3‐week period throughout the growth season, followed by a 3‐week incubation period in a growth chamber to promote symptom expression. In second year seedlings the infection periods in May–June had a similar effect as regards disease outbreak and roughly half of the successful infections occurred during these periods. However, the first year seedlings were more severely diseased after the infection period in late July/early August, although the inoculum density was lower during this time compared with the period in late June/early July. This result is consistent with earlier studies. The effect of age and growth phase of the seedlings, temperature sum and the number of conidia on the disease occurrence is discussed and compared with the results of earlier studies in which artificial inoculation was employed.     

Exposure to a pine pathogen enhances growth and disease resistance in Pinus radiata seedlings

Most studies of Fusarium circinatum, the cause of pitch canker in pines, have focused on its activity as a pathogen. However, recent findings indicate that this fungus can colonize roots of Pinus radiata without inducing symptoms. Contrary to expectations, this study revealed that seedlings grown in infested sand grew more rapidly than seedlings not exposed to F. circinatum, based on root and shoot biomass, with modifications to root system architecture, including increased mycorrhizal root development. These effects were dependent on inoculum density and duration following growth in infested rooting medium. Plants exposed to F. circinatum expressed elevated resistance to stem infections, which significantly decreased the incidence of mortality; as above, effects were dependent on inoculum density. Resistance to stem infections was also enhanced in seedlings that emerged through infested litter, as occurs in native stands. Beneficial to neutral interactions of F. circinatum with its host suggest that the life history of this fungus may be more complex than previously recognized, with activities similar to non‐pathogenic endophytes. The potential for non‐lethal infections by F. circinatum to induce resistance in seedlings may influence dynamics of stand establishment. Overall, these results indicate that pathogenic organisms with asymptomatic states may have cryptic ecological functions that extend beyond the impacts of disease.     

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.     

Effects of inoculum density of pinewood nematode on the development of pine wilt disease in Japanese black pine seedlings pretreated with simulated acid rain

Six‐month‐old Japanese black pine seedlings (Pinus thunbergii) were exposed to simulated acid rain (SAR) at pH 3 and 2 three times a week. After treatment for 2 months, the seedlings were inoculated with a virulent isolate (S10) of the pinewood nematode (Bursaphelenchusxylophilus), at three inoculum levels (P_i = 50, 160 or 500 nematodes per seedling). In seedlings inoculated with 500 nematodes, both population growth of nematodes and disease development were accelerated by pretreatment with SAR at pH 3 or 2. In seedlings inoculated with 50 nematodes, population growth of the nematodes was suppressed and more time was needed for seedlings to die when pretreated with pH 3 SAR. This suggests that exposure to pH 3 SAR increased not only the progress of mortality, but also simultaneously enhanced the tolerance limit of the seedlings to the pinewood nematode – the critical value of physiological burden (represented as a product of time and initial nematode population) necessary to kill a seedling. Exposure to pH 2 SAR accelerated nematode reproduction in seedlings and increased seedling mortality irrespective of the number of nematodes inoculated.     

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.     

Diplodia twig canker (Diplodia gallae) of northern red oak (Quercus rubra) in the northeastern United States

The 2015–2018 outbreak of spongy moth (Lymantria dispar) in southern New England initiated a severe oak decline and mortality event. While defoliation was a primary driver, increased secondary pest and pathogen activity contributed to decline and death. Following this large defoliation event, Diplodia was frequently recovered from northern red oaks (Quercus rubra) with serious outbreaks of twig cankering. Given the many recent reports of Diplodia corticola on oak in eastern North America, it was presumed to be the causal agent. To confirm, a limited survey was conducted from five states in the region (Maine, Massachusetts, New Hampshire, New York, and Vermont). Based on ITS, tef1 and tub2 sequences generated from 28 isolates collected primarily from northern red oak, Diplodia gallae, two potentially novel Diplodia species and Diplodia sapinea were identified. Surprisingly, D. corticola was not found in this study. ITS sequences alone failed to discriminate among isolates of D. gallae and D. corticola, creating uncertainty over previous reports of D. corticola in eastern North America. Only a combined ITS + tef1 dataset successfully distinguished D. gallae and D. corticola along with two other closely related species that also occur on oak (Diplodia quercicola and Diplodia quercivora). Additional cankering and endophytic fungi (Coryneum, Dendrostoma, Gnomoniopsis, Pestalotiopsis and Tubakia) were also found on symptomatic oaks in the region. Identification of Diplodia isolates from non-Quercus hosts also detected Diplodia neojuniperi on Juniperus chinensis and Microbiota decussata, which has not been reported previously in North America.     

Photosynthetic acclimation of shade-grown red pine (Pinus resinosa Ait.) seedlings to a high light environment

Photosynthetic light acclimation in red pine (Pinus resinosa Ait.) seedlings was examined in a greenhouse study to better understand the physiological response of this species to increased light intensity following release from competition. Seedlings grown in a high (HL), medium (ML) or low (LL) light environment for 12 weeks were transferred to high light. Gas exchange and chlorophyll fluorescence of ML and LL seedlings were measured prior to and following transfer and compared with the HL control treatment. Photosynthetic characteristics were related to initial light treatment and time after transfer. Acclimation of gas exchange features to high light in shade formed ML and LL foliage was relatively rapid, with similar values among light treatments within 57 days of transfer. Acclimation of net photosynthetic rate was similar in ML and LL seedlings, and was associated primarily with increased mesophyll conductance to CO₂. The ratio of variable to maximal chlorophyll fluorescence (F_v/F_m) decreased initially after transfer, especially in LL seedlings, but recovered to normal values after 57 days. Red pine seedlings appear to be well adapted for photosynthetic acclimation to high light intensity, consistent with that reported for other early successional tree species.     

Drought-dependent responses of photosynthesis,transpiration and water use efficiency of Japanese cypress and Japanese red pine seedlings

This study was conducted to investigate the potential for modifying drought tolerance of Japanese cypress (Chamaecyparis obtusa Endl.) and Japanese red pine (Pinus densiflora Sieb. et Zucc.). Three-year-old seedlings were controlled for five-months at three different soil water potentials ({ie73-1}). Japanese cypress exposed to high {ie73-2} was able to maintain higher photosynthesis (Ph_n), transpiration (Tr) and stomatal conductance to H₂O (gH₂O) in comparison to low {ie73-3} pretreatments, however, there was no significant difference in Ph_n for Japanese red pine. Soil water potential at the threshold from the maximum to limited Ph_n was higher in high {ie73-4} pretreatments than in low {ie73-5} pretreatments. Net photosynthesis, Tr and gH₂O decreased more rapidly in high {ie73-6} pretreatments than in low {ie73-7} pretreatments. Transpiration decreased more significantly than Ph_n, thus, resulted in increased water use efficiency. All these factors are likely to result in significant improvements in the drought tolerance. Japanese red pine seems more drought-tolerant than Japanese cypress. Japanese cypress is suitable to soil of −0.05 MPa water potential, and Japanese red pine is suitable to −0.16 MPa and even dryer soils.     

Effects of contrasting fertilization and moisture regimes on biomass,nutrients, and water relations of container grown red pine seedlings

Red pine seedlings were grown for 16 weeks under contrasting fertilizat (conventional, exponential) and moisture (wet, moist, dry) regimes to assess preconditioning effects of treatments on biomass production, nutrient uptake and allocation, and water relations. Growth, nutrient status, and water relations were affected more by moisture availability than by fertilization regime. Exponential fertilization under limited irrigation lowered shoot/root mass ratio, increased root nutrient reserves, and enhanced drought avoidance compared to conventional fertilization regimes. Drought treatments decreased nutrient uptake in the shoots of both fertilization regimes by 24%, but increased nutrient accumulation in the roots by 39% in the exponential regime compared to 17% in the conventional. These results may explain improved outplanting performance noted for exponentially fertilized container stock.     

Field survival of containerized red and jack pine seedlings inoculated with mycelial slurries of ectomycorrhizal fungi

Red pine and jack pine seedlings growing in styroblocks were inoculated 8 wk after sowing with mycelium/agar slurries of 3 mycorrhizal fungi (Laccaria bicolor, Scleroderma citrinum, and an unidentified basidiomycete), and one suspected mycorrhizal fungus (Cantharellula umbonata). Seedlings inoculated with L. bicolor developed mycorrhizae earlier and in greater numbers than the other inoculation treatments, with red pine out-performing jack pine in both respects. At 34 wk following sowing, seedlings were outplanted on a cleared xeric site in Baraga Co., in Michigan's Upper Peninsula. Seedlings inoculated with C. umbonata failed to form mycorrhizae and were not outplanted. Inoculation treatments did not affect shoot or root weight at outplanting. Red pine inoculated with L. bicolor averaged 21% and 19% greater survival compared with control seedlings after one and two years in the field, respectively. Other inoculation treatments failed to increase seedling survival for either tree species. Jack pine demonstrated higher overall survival than did red pine for both years in all corresponding inoculation treatments.     

Systemic effects of Heterobasidion annosum s.s. infection on severity of Diplodia pinea tip blight and terpenoid metabolism in Italian stone pine (Pinus pinea)

Three-year-old seedlings of Pinus pinea L. were inoculated near the stem base with one of two Heterobasidion annosum (Fr.) Bref. sensu stricto (s.s.) strains belonging to two populations: the North American P-group (NAm-P) and the European P-group (Eur-P). The NAm-P strain caused smaller H. annosum stem lesions than the Eur-P strain. Three weeks after the stem inoculations with H. annosum, apical shoots were inoculated with Diplodia pinea (Desmaz.) J. Kick. Basal stem infection with H. annosum resulted in D. pinea causing longer necrotic lesions in the shoots, indicating systemic induced susceptibility (SIS) to this shoot blight pathogen. Furthermore, stem induction with the NAm-P strain resulted in higher susceptibility to D. pinea than stem induction with the Eur-P strain. Total terpene accumulation was suppressed by about 50% in the shoots under attack by D. pinea when seedlings were induced with H. annosum. Total terpene concentration in shoots inoculated with D. pinea was negatively correlated with lesion size, both overall and by stem treatment. Stem base inoculation with H. annosum induced whole-plant changes in ter-pe-noid profiles, but these were not associated with the SIS phenotype. We discuss our findings on modulation of systemic response of P. pinea to fungal attack in the context of tripartite ecological interactions.