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.     

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.     

Excised shoots of top-pruned red pine seedlings,a source of inoculum of the Diplodia blight pathogen

Diplodia pinea causes shoot blight and collar rot diseases of pines in forest tree nurseries and sporulates on colonized seedling needles and stems. In late summer 2005, pycnidia of D. pinea were observed on shoots that had been excised by top pruning red pine seedlings earlier that summer during the third season of growth. This observation prompted surveys to determine the incidence and abundance of D. pinea conidia on excised shoots. At each of two nurseries, excised shoots were collected from the seedling canopy and adjacent alleyway soil in two subplots in each of five beds (plots). Excised shoots from both nurseries bore pycnidia with conidia of D. pinea. A water washing and filtration technique was used to quantify D. pinea conidia extracted from these shoots. Excised shoots collected from the seedling canopy yielded more D. pinea conidia than shoots collected from adjacent alleyway soil. Collection and removal of excised shoots resulting from top pruning of pine nursery seedlings should be considered as a means of reducing inoculum in areas where D. pinea is present.     

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.     

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.     

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.     

Histology of Diplodia pinea in diseased and latently infected Pinus nigra shoots

Diplodia tip blight is a serious disease of >30 conifer species worldwide. Symptoms are particularly severe on non‐native, two‐needled Pinus species, and typically include stunted, necrotic needles and shoots and a general decline of the tree. Latent Diplodia pinea infections occur in current‐year shoots of some symptomless pines, and in some apparently healthy current‐year shoots of diseased pines. Latent infections also occur in symptomless terminal buds. A histological approach was used to investigate the nature of latent infections in shoot stems and terminal buds of landscape Pinus nigra. Fungal colonization was compared in healthy, diseased and latently infected tissues. A nested‐PCR technique that is specific for D. pinea was used to differentiate latently infected tissues from uninfected ones. Latent D. pinea infections were localized in the outer stem cortex, usually in the vicinity of needle scales at leaf axes. In contrast, pathogenic D. pinea infections were characterized by fungal colonization throughout the shoot stem tissues, even very early in symptom development. The presence of necrophylatic periderms in two of the latently infected samples suggests that host defences play a role in the production and maintenance of latent stem infections. Latent infections of terminal buds appeared to originate from the distal bud scales of axillary buds in the terminal bud cluster, and not from the subtending shoot. Fungal tissues were never observed inside asymptomatic, PCR‐negative shoots.     

Resistance levels of Spanish conifers against Fusarium circinatum and Diplodia pinea

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

Diplodia scrobiculata found in the southern hemisphere

Diplodia scrobiculata, a latent pathogen of Pinus spp. and other conifers with a limited distribution in the United States, Mexico and southern Europe, has not been reported previously in the southern hemisphere. This is unlike its close relative Diplodia pinea that is found in most parts of the world where pines are native or have been introduced. During an intensive D. pinea survey conducted in Pinus patula plantations in eastern parts of South Africa, a small number of isolates atypical of D. pinea were found. Morphological studies and DNA sequence comparisons showed that these isolates represent D. scrobiculata. Microsatellite analyses suggest that the South African isolates of D. scrobiculata might have originated from California. Pathogenicity tests showed that some of the D. scrobiculata isolates were as pathogenic as those of D. pinea on Pinus radiata and Pinus elliottii.     

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.     

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

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

Leptoglossus occidentalis and Diplodia pinea: a new insect‐fungus association in Mediterranean forests

Leptoglossus occidentalis, an insect native to North America, was inadvertently introduced into Italy about 1999. The insect damages the cones of conifer trees, especially Pinus pinea (Italian Stone pine). Pinus pinea is also affected by Diplodia pinea, a fungus native to Italy, which is becoming an increasing threat because pine trees are becoming more susceptible to it as a consequence of global warming. Because the insect and the fungus both have the pine cones as a common habitat, a possible interaction between them has been postulated. The aim of this study was to ascertain whether L. occidentalis and D. pinea interact on P. pinea cones. The interaction was studied using real‐time PCR on a group of naturally infected insects collected from a forest, and a group raised in the laboratory and artificially inoculated with D. pinea conidia. Molecular analysis showed that D. pinea DNA occurred on both naturally infected and inoculated insects, but with significant differences between the two groups. The rapid and sensitive molecular technique made it possible to detect D. pinea DNA on the bodies of the insects, and to show that the native D. pinea occurred on the exotic insect.     

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.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.     

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.     

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.     

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.