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.     

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.     

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.     

Organ-dependent induction of systemic resistance and systemic susceptibility in Pinus nigra inoculated with Sphaeropsis sapinea and Diplodia scrobiculata

Systemic induced resistance (SIR) is a well-known host defense mechanism against pathogen attack in herbaceous plants, but SIR has only recently been documented in conifers. We tested if inoculation of Austrian pine (Pinus nigra Arnold) with Sphaeropsis sapinea (Fr.:Fr.) Dyko and Sutton or Diplodia scrobiculata de Wet, Slippers and Wingfield results in SIR or systemic induced susceptibility (SIS) to subsequent colonization by S. sapinea. Induction at the stem base resulted in significant (P < 0.01) SIR in the upper stem, and induction in the upper stem resulted in significant (P < 0.05) SIR at the stem base, indicating that SIR is bidirectional in Austrian pine. However, inoculation at the stem base resulted in significant (P < 0.01) SIS in shoot tips, demonstrating that, in the same host species, the expression of resistance can be organ-dependent, resulting in either SIR or SIS depending on the site of challenge infection. Systemic induced resistance in the stem was associated with induced lignification, supporting a potential role for this defense mechanism in disease resistance. Systemic induced susceptibility has been documented before, but this is the first demonstration of organ-dependent expression of both SIR and SIS in a tree or any other plant.     

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.     

Host specificity of co‐infecting Botryosphaeriaceae on ornamental and forest trees in the Western Balkans

The Botryosphaeriaceae is a diverse family of endophytes and fungal pathogens of mainly woody plants. We considered the host range and distribution of these fungi by sampling diseased ornamental and forest trees and shrubs in Serbia, Montenegro, Bosnia and Herzegovina, spanning a Mediterranean and a Continental climatic region. In total, ten Botryosphaeriaceae species were identified in the Western Balkans and with the exception of Sphaeropsis visci and Phaeobotryon cupressi, which occurred on one host, all the species had a broader host range. Phaeobotryon cupressi was found only in the Mediterranean region and S. visci, Dothiorella sp., Dothiorella sarmentorum and Diplodia seriata were present only in the Continental region. Pathogenicity tests were conducted on a variety of hosts from which the Botryosphaeriaceae species were isolated. These included leaves and/or stems of seedlings of 21 hosts, and cut leaves and/or branches of six hosts. Moreover, stems of seedlings of Chamaecyparis lawsoniana, Cedrus deodara, Picea omorika, Pinus patula and Eucalyptus grandis were inoculated as hosts from which some or all of the Botryosphaeriaceae species used for inoculation were not isolated. Inoculations showed that the majority of these fungi could also co‐infect hosts other than those from which they were isolated. The results suggest that most of the species have broad host ranges and can potentially cause disease on a broad range of tree species under certain conditions.     

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.     

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.     

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.     

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.     

松球壳孢菌菌落及孢子形态学对比研究

通过79个辽宁松球壳孢菌菌株与4个A型和B型标准菌株、5个美国菌株在菌落和分生孢子形态方面的对比研究，发现辽宁的菌株在形态上基本一致。辽宁菌株与美国的类型A相近，主要表现在孢子表面平滑，通常无隔或有1个隔，在成品PDA培养基上产生绒毛状、白色至灰色菌落。不同之处在于，辽宁菌株在自制PDA培养基上形成贴在培养基表面的菌落——与类型B相似，分生孢子长度介于A型和B型之间或与后者相近。5个美国菌株与类型A一致。同时发现，培养基的来源对菌落形态研究非常重要；光照使菌落气生菌丝有所减少且出现绿色；培养基和寄主种类对孢子大小有影响。     

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.     

Antagonism of Acremonium mucronatum towards Diplodia mutila in tests in vitro and in situ

Tests in vitro and in situ showed that both mycelial and conidial suspensions of Acremonium mucronatum were antagonistic for Diplodia mutila, a common pathogen on the apical twigs of declining Quercus cerris, Quercus frainetto, Quercus pubescens and Quercus robur. Antagonism was greater with the mycelium than with the conidial suspensions, and was strongest at a test temperature of 20°C. The results suggest that pathogenicity of D. mutila on declining trees may be significantly reduced by the presence of A. mucronatum.     

我国西部木材市场近期出现新变化

近年来我国西部木材市场经过了几次较大的波动,特别是2004-2005年期间,由于国家提出西部大开发的战略及青藏铁路、南水北调、西气东输、西电东送等一系列国家重点工程的启动、修建,引发和牵动了包括木材市场在内的诸多建材的需求强劲上扬。这一大好的趋势,持续的时间大约在一年之久,甚至在一段时间内,不少木材业界人士怀疑,莫非国内木材市场的中心果真从东部沿海转移到了西部地区？     

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.     

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.     

The Macedonian Pine, (Pinus peuce Grisebach) in the Balkans and Great Britain

Macedonian pine is little known in Britain as a forest tree.Information comes mainly from its native range in the Balkanmountains, and from specimen trees and young forest plots inBritain. Following a visit in 1982 to Yugoslavia and Bulgaria,the author describes the site conditions and ecological relationsof this species to Scots pine, Norway spruce and Silver fir.Early growth is characteristically slow, even on favourablesites, though it improves after 6–10 years and basal areaincrement may later exceed that of other pines. Growth and behaviourin British stands (mainly 20 years old, but a few older ones)is recorded. Stands of P. peuce are typically healthy and haveexcellent form. Insect and fungal damage is minimal. Early timbertests show specific gravity and strength are low, but the woodis very stable in drying and could have a use in joinery. Seedfrom selected stands will now be used for provenance testing.