Diplodia corticola and Phytophthora cinnamomi: the main pathogens involved in holm oak decline on Caprera Island (Italy)

Since 2008, severe and widespread tree decline and mortality has been observed at the main growing Quercus ilex L. (holm oak) forest on Caprera Island, Italy. To clarify the symptomatology and aetiology of this phenomenon, field surveys and isolations from symptomatic trees were carried out in summer 2010. Affected trees exhibited crown thinning, branch dieback, sunken cankers, epicormic shoots, exudates on branches and trunk, root losses and sudden death symptoms. Four fungal species belonging to Botryosphaeriaceae family, namely Botryosphaeria dothidea, Diplodia corticola, D. seriata and Neofusicoccum parvum, were isolated from cankers on trunk and branches, whereas three species of Phytophthora, namely P. cinnamomi, P. cryptogea and P. gonapodyides, were isolated from fine roots and rhizosphere soil samples. Isolates were identified using both morphological analysis and DNA‐based techniques. Pathogenicity trials on holm oak seedlings showed that all the isolated species are pathogenic. D. corticola proved to be the most aggressive species. Our results provide the first evidence for a combined involvement of D. corticola and P. cinnamomi in the aetiology of holm oak decline in Italy and suggest that these pathogens are not only important contributing factors in the onset of long‐term tree decline, but also may cause the rapid devastation of extensive oak ecosystems.     

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.     

Identification and characterization of fungi associated with internal wood lesions and decline disease of willow and poplar trees in Iran

During the study of fungal trunk pathogens associated with urban trees decline in Shiraz (Iran), a serious decline of willow and poplar trees was observed. Therefore, an investigation was conducted on these trees in some areas of this city during spring and summer 2012 and 2013, to determine the main fungal trunk pathogens associated with these ornamental plants. Plant materials were collected from trees exhibiting disease symptoms such as yellowing, shoot canker, shoot dieback, defoliation and internal wood necrosis and decayed wood. Fungal isolations were made from discoloured or decayed wood tissue onto 2% malt extract agar (MEA) amended with streptomycin sulphate. Nine species, Fomes fomentarius, Diplodia seriata, Lasiodiplodia theobromae, Dothiorella sarmentorum, Neoscytalidium hyalinum, Diatrype whitmanensis, Phaeoacremonium rubrigenum, P. aleophilum and P. parasiticum, were identified based on morphology and DNA sequence comparisons. Pathogenicity tests were performed on detached shoots of willow and poplar trees under greenhouse conditions. Lasiodiplodia theobromae caused the longest lesions on willow. On poplar shoots, the longest lesions were caused by P. parasiticum. Diplodia seriata produced the smallest lesions on both woody hosts. First reports from willow wood include P. parasiticum, P. rubrigenum, D. whitmanensis, L. theobromae, D. seriata and N. hyalinum, while new reports from poplar wood include P. parasiticum and Do. sarmentorum. Based on our knowledge, this is also the first report of D. whitmanensis in Iran.     

Genetic uniformity characterizes the invasive spread of Neofusicoccum parvum and Diplodia sapinea in the Western Balkans

In the past decade, trees and shrubs in the Western Balkans region have been damaged by canker and die‐back disease caused by Botryosphaeriaceae species. These pathogens include Neofusicoccum parvum and Diplodia sapinea. In this study, we determine genetic diversity and structure between populations of N. parvum and D. sapinea from Serbia and Montenegro (Western Balkans) using DNA sequence data of the internal transcribed spacer rDNA, translation elongation factor 1‐alpha, β‐tubulin‐2 and microsatellite markers. The relationship of both pathogens was compared for populations from the Continental (CR) and Mediterranean (MR) regions and for isolates of D. sapinea from Cedrus spp. and Pinus spp. Neofusicoccum parvum and D. sapinea were shown to have a low gene and genotypic diversity across the regions and hosts. All genotypes of D.  sapinea found on Pinus spp. were also present on Cedrus spp. The CR and MR populations of both species were found to be only slightly separated from one another by a geographical barrier. Low genetic diversity and dominance of N. parvum and D. sapinea on non‐native trees suggests that these species have most likely been introduced into Western Balkans, possibly through the movement of infected plants.     

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.     

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.     

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.     

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.     

Fungal pathogens associated with twig canker of shrub species in Tunisia: Considering the effect of the factors correlated

Decline phenomena of shrub species such as Quercus coccifera and Retama raetam have occurred throughout Tunisian forests since 2012. These evergreen shrubs have long been regarded for their medicinal and ecological interests. Therefore, their preservation as valuable forest resources is of great interest. However, information regarding aetiology of this disease is still scarce. Hence, the aim of this study was to identify and characterize the causal agents associated with disease symptoms in two Tunisian forests. Thirty-eight isolates were obtained from symptomatic Q. coccifera and R. raetam twigs. Morphological characterization and phylogenetic analysis of the internal transcribed spacer (ITS) region of the nuclear ribosomal RNA gene cluster and partial sequence of the translation elongation factor 1-alpha gene (tef1-α) allowed the identification of three Diplodia species namely Diplodia africana, D. seriata and D. pseudoseriata. Our findings revealed that the incidence of Diplodia species was significantly correlated to the altitude, the temperature and the rainfall. Pathogenicity test showed that all Diplodia isolates are pathogenic. However, D. africana revealed to be the most aggressive species toward R. raetam. These findings were the first record of D. seriata as fungal pathogen associated with Q. coccifera dieback and D. pseudoseriata and D. africana on R. raetam in Tunisia.     

Impact of Botryosphaeria,Diplodia and Neofusicoccum species on two Eucalyptus species and a hybrid: From pathogenicity to physiological performance

Species of Botryosphaeriaceae are associated with canker and dieback of Eucalyptus spp. worldwide, but little is known about their effect on the host physiology. The aim of this study was to evaluate the impact of Botryosphaeriaceae isolates from nine species in three genera (Botryosphaeria, Diplodia and Neofusicoccum), previously isolated from eucalypts, on three different Eucalyptus hosts (seedlings of E. nitens, cuttings of E. globulusand of E. globulus× E. cypellocarpa). An approach combining standard pathogenicity trials with evaluation of plant morpho‐physiological parameters was used. The size of the lesions produced revealed differences in fungal aggressiveness and host susceptibility. Isolates of Neofusicoccum kwambonambienseand Diplodia corticolawere the most aggressive, while Botryosphaeria dothidea and Diplodia seriataisolates were the least aggressive. In general, hybrid E. globulus× E. cypellocarpa plants developed smaller lesions, followed by E. nitens and E. globulus. Eucalyptus nitensplants showed minimal modifications on the morpho‐physiological profile when infected, although more severe symptoms and mortality were observed. This is probably due to a more variable genetic background of the plants. However, in general, no direct association between putative fungal aggressiveness and plant physiological disorders could be found. Results suggested that under optimal growth conditions plants manage to cope with pathogen attack and maintain their physiological performance.     

Virulence and double‐stranded RNA in Sphaeropsis sapinea

Forty wildtype isolates of Sphaeropsis sapinea were grouped into the morphotypes A and B based on previously defined differences in cultural and morphological criteria as well as restriction sites for Dde I and Bst UI endonucleases in nuclear ribosomal DNA amplicons. Thirteen of 20 type A isolates and nine of 20 type B isolates contained detectable dsRNA (55%) of different molecular weight and size. dsRNA was transmitted into conidia at a frequency of 71–100%. By selecting single conidia, dsRNA‐free subcultures were obtained from six of 22 isolates containing dsRNA. Pathogenicity tests on expanding buds of landscape trees of three species of Pinus showed highly significant statistical interactions between isolate virulence, Pinus species, and year. Pine species‐year had a profound impact on virulence. The pattern in the interactions was revealed by principal component analysis of the interaction sums of squares of the anova (Additive Main Effects and Multiplicative Interaction; AMMI). Pinus sylvestris was highly interactive in its susceptibility to S. sapinea with seasonal effects. P. nigra and P. resinosa were more stable. The interactivity analysis was used to apportion interaction to specific isolates to improve the accuracy of the estimates of virulence. Estimates of the relative virulence of isolates were predicted over five different Pinus species‐years. Isolates were ranked in virulence and interactivity using the AMMI model. This model permitted mean separation tests of the relative virulence among isolates over the combined Pinus species‐years. One isolate was identified as potentially having dsRNA‐mediated hypovirulence based on the significantly greater virulence of its isogenic, dsRNA‐free subculture, as expressed over the three Pinus species and 2 years. Type A isolates containing dsRNA ranged from stable to highly interactive and from low to high in virulence. Type B isolates containing dsRNA were similar in interactivity but virulence ranged from avirulent to moderate, seldom exceeding the mean for S. sapinea. dsRNA‐free isogenic subcultures tended not to express higher virulence than their dsRNA‐containing parent strains but often changed in interactivity. Therefore, in one year a dsRNA‐free subculture might be more virulent than its dsRNA‐containing parent. In another year the dsRNA‐free subculture might be less virulent.     

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.     

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.     

Occurrence of Diplodia corticola,including new oak host records,in Wisconsin,USA

In the United States, diseased oaks (Quercus species) exhibit tip blight, branch and stem cankers, and dieback often attributed to Diplodia species or related fungi. Emergence of Diplodia corticola as a pathogen of European oaks, and reports of this fungus in the eastern and western United States, prompted re‐examination of strains from Wisconsin. These had been obtained in the late 1990s and early 2000s and previously identified only as Diplodia species. Nuclear ribosomal ITS sequences were obtained from the Wisconsin strains and analysed with other sequences from GenBank. Wisconsin strains confirmed as D. corticola were from northern red oak (Q. rubra), black oak (Q. velutina), white oak (Q. alba) and bur oak (Q. macrocarpa). Other strains from oaks in Wisconsin were D. mutila and D. seriata. Wound inoculation of northern red, white and bur oak seedlings with D. corticola in a greenhouse resulted in shoot death and stem lesions, from which the pathogen was reisolated. We conclude that D. corticola has been present in the northcentral United States for at least two decades and report two previously unrecognized hosts of this pathogen: white oak and bur oak. The roles of D. corticola, related fungi and influences of other environmental factors in deterioration of oak health in North America merit additional investigation.     

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.     

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.     

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.