Sphaeropsis Sapinea Research Articles

Interaction between Diplodia pinea or Diplodia scrobiculata and fungal endophytes isolated from pine shoots

Four endophytes isolated from healthy shoots of adult red pine ( Pinus resinosa Aiton) or jack pine ( Pinus banksiana Lamb.) trees were tested as potential biocontrol agents of the conifer shoot blight and canker pathogens Diplodia pinea (Desmaz.) J. Kickx fil. and Diplodia scrobiculata J. de Wet, B. Slippers & M.J. Wingfield. The effect of the endophytes on the germination of Diplodia species conidia and on the symptom severity caused by these pathogens on P. banksiana seedlings under greenhouse conditions was evaluated. Aggressiveness of each organism also was determined by means of single inoculations on jack pine seedlings. The results indicated that the endophyte Trichoderma atroviride P. Karst. showed a clear antagonism to Diplodia species. The culture filtrate of this fungus was able to reduce spore germination of D. scrobiculata. Furthermore, it was able to inhibit the D. pinea invasion when both organisms were inoculated in the same seedling. The observed reduction in the symptom severity on jack pine seedlings might be due to parasitism and (or) production of inhibitory compounds. Therefore, T. atroviride might deserve further investigations for its use as biocontrol agent against D. pinea.

Seleção precoce de progênies de <i>Pinus radiata </i>a <i>Sphaeropsis sapinea</i>

http://dx.doi.org/10.5902/198050985734Sphaeropsis sapinea é um importante patógeno para Pinus radiata, causando seca de ponteiros e morte de árvores, em plantios comerciais. Este estudo teve como objetivo selecionar progênies de Pinus radiata resistentes ao patógeno. Mudas de 16 progênies de Pinus radiata foram inoculadas com três isolados monospóricos do patógeno, em condição de casa de vegetação. Foram avaliados o comprimento das lesões e o grau de secamento dos ponteiros. Os resultados indicaram que a metodologia de seleção precoce foi adequada para selecionar progênies de Pinus radiata resistentes ao Sphaeropsis sapinea. A herdabilidade média das progênies variou de acordo com o isolado testado e o critério de seleção utilizado.

Frequency of virus in some Diplodia pinea and Gremmeniella abietina isolates originated from Turkey

Diplodia pinea and Gremmeniella abietina are common pathogens causing shoot blight and dieback of pine all over the world. D. pinea is one of the main causal agents of shoot blight of Calabrian pines in the Mediterranean countries including Turkey. G. abietina has been recently observed on saplings and seedlings of Pinus nigra, which remain under snow cover during winter dormancy in Dedegul Mountain in the Mediterranean Region of Turkey. The presence of viruses in fungi has been known for many years. An accumulating number of cloned and sequenced viral genomes have enabled us to detect virus in increasing number of fungal species in the recent years. D. pinea and G. abietina are known to contain members of the virus families Narnaviridae, Totiviridae and Partitiviridae, which can infect single fungal isolates. Viral dispersal in fungi mainly occurs via anastomosis. Some Diplodia and G. abietina isolates have different characteristics, such as reduced virulence and growth rate, lack of pigmentation, altered colony morphology, and reduction in conidial production due to presence of viral particles. In this study, 18 D. pinea and 6 G. abietina isolates were investigated for the presence of dsRNA. Double-stranded RNA was isolated using a commercial RNA extraction kit and visualized in agarose gel electrophoresis. Isolates containing dsRNA were also investigated for their in vitro growth rate and ability to produce conidia. Three (50%) G. abietina and ten (56%) D. pinea isolates contained dsRNA that had an approximate molecular size of 1.6 kb. Key words: Scleroderris canker, calabrian pine, dsRNA.

Patogenicidade de Sphaeropsis sapinea em árvores jovens de Araucaria angustifolia

Sphaeropsis sapinea é um patógeno que causa seca de ponteiros e morte de árvores em plantios comerciais de coníferas do mundo. No Brasil, a literatura apresenta este fungo em mudas e toras de Araucaria angustifolia após a colheita, sem relatos de seca de ramos e de ponteiros. Este estudo teve como objetivo verificar a patogenicidade de S. sapinea em Araucaria angustifolia. O postulado de Koch foi realizado em 25 árvores com 2 anos de idade de A. angustifolia. Discos de micélio-ágar de um isolado agressivo obtido de Pinus taeda, retirado de culturas com 10 dias de idade, foram inoculados em ramos injuriados. Três ramos foram inoculados com o fungo, deixando-se um ramo como testemunha, por planta. Todas as plantas foram mantidas em casa de vegetação por 30 dias. Ramos com e sem sintomas foram colocados em câmara úmida, sob iluminação, para reisolar o patógeno. Oito árvores inoculadas apresentaram sintomas similares aos provocados pelo S. sapinea em pínus. O fungo foi reisolado de 68 % das árvores inoculadas. Confirmou-se a seca de ramos causado por S. sapinea em árvores jovens de araucária.

The influence of Amylostereum areolatum diversity and competitive interactions on the fitness of the Sirex parasitic nematode Deladenus siricidicola

The Sirex noctilio (woodwasp)–Amylostereum areolatum (fungus) complex has caused substantial losses to pine industries in its introduced range. The nematode Deladenus siricidicola that parasitizes S. noctilio and feeds on A. areolatum is widely used as a biological control agent for S. noctilio, but not with consistent success. This variable success could be due to factors that influence the feeding and reproductive ability of the nematode on A. areolatum. We test two main hypotheses that emerge from this prediction. First, we compared the survival of D. siricidicola on the South African field strain and the Australian laboratory strain of A. areolatum, to examine a possible incompatibility between nematode and fungal strain. Second, we examined the competitive interactions of these two A. areolatum strains with two common sapstain fungi, Diplodia pinea and Ophiostoma ips, that occur in S. noctilio infested trees in South Africa. The effect of water potential on the outcome of these fungal interactions was also considered. The data showed that D. siricidicola survives at comparable levels on the two A. areolatum strains. Water potential of the media significantly influenced growth of the fungi and their ability to capture host resource in competitive interactions. D. pinea competed increasingly better against A. areolatum with decreasing water potential. The results suggest that competitive interactions between A. areolatum and sapstain fungi could negatively influence the success of D. siricidicola, especially under conditions of lowered water potential.

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

SummaryLeptoglossus 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.

Diverse sources of infection and cryptic recombination revealed in South African Diplodia pinea populations

This study considers the population diversity and structure of Diplodia pinea in South Africa at different spatial scales from single trees to plantations, as well as comparing infections on healthy and diseased trees. A total of 236 isolates were characterized using 13 microsatellite markers. Analysis of these markers confirmed previous results that D. pinea has a high level of gene and genotypic diversity in South Africa, with the latter values ranging from 6% to 68% for the different plantations. The data also reflect a fungus with randomly associated alleles in populations at local plantation scales and for the population as a whole. These results suggest that recombination is occurring in D. pinea and that it most likely has a cryptic sexual state. The study also reveals the sources of endophytic infection and stress related disease out-breaks as diverse infections that have occurred over a long time period. In contrast, wound-associated die-back appears to be caused by clones of the pathogen occurring in narrow time frames.

High levels of genetic diversity and cryptic recombination is widespread in introduced Diplodia pinea populations

Introduced populations of organisms typically have reduced diversity compared to those that are native. It is, therefore, unusual that introduced populations of the fungal tree pathogen Diplodia pinea have been shown to have high levels of genetic diversity, even surpassing diversity in some native regions. This is thought to be due to multiple introductions over time or the existence of a cryptic and yet undiscovered sexual cycle. In this study, we consider whether populations of D. pinea in Southern Hemisphere countries have similar patterns of diversity, share some level of genetic identity and how they might be influenced by sexual recombination. A total of 173 isolates from Argentina, Australia, Ethiopia and South Africa were characterized using 12 microsatellite markers. The results show that all these populations have high gene and genotype diversities, with the Australian population having the lowest diversity. Very few private alleles were found, suggesting that isolates from different countries might share a source of introduction. However, based on allele distribution and frequency, each of the populations appeared to be evolving independently. The results showed that in all but the Australian population, alleles are randomly associated, suggesting that widespread sexual recombination has influenced population structure.

METODOLOGIA DE AVALIAÇÃO DA AGRESSIVIDADE DE ISOLADOS DE Sphaeropsis sapinea A Pinus taeda

Sphaeropsis sapinea é conhecido como importante patógeno em Pinus, causando seca de ponteiros e morte de árvores em plantios comerciais. No Brasil, o primeiro relato da ação de S. sapinea ocorreu na década de 1940, durante introdução de Pinus radiata no estado de São Paulo, dizimando totalmente os plantios. Com o objetivo de selecionar material genético resistente a essa doença no Brasil, desenvolveu-se uma metodologia para avaliar a agressividade de isolados de S. sapinea em frutos de maçã e Pinus taeda. Três ambientes (estufas com diferentes regimes de irrigação), dois métodos de inoculação (com e sem injúria) e três métodos de reisolamento do fungo (meio BDA, câmara úmida em saco plástico e em gerbox úmido) foram testados. Resultados mostraram diferenças significativas (p<0,05)na agressividade dos isolados em frutos de maçã e em mudas de P. taeda. O melhor método de reisolamento foi a câmara úmida e o melhor ambiente para a incubação de mudas de P. taeda foi em estufa coberta com lona plástica, com duas aspersões diárias de 4 minutos. Outro resultado foi que injúria é necessária para inoculação de S. sapinea. Os resultados da caracterização mostraram diferenças significativas na agressividade e diversidade genética do patógeno em todos os hospedeiros testados.Palavras-chave: Fungo; inoculação; melhoramento genético; Pinus.AbstractEvaluation methodology of the aggressiveness of isolated of Sphaeropsis sapinea on Pinus taeda. Sphaeropsis sapineais known as an important pathogen of Pinus, causing tip blight and death of trees in commercial plantings. In Brazil, the first report of this disease was done in the 1940s, during introduction of Pinus radiata in São Paulo State, when these plantations were destroyed by S sapinea. In order to select material resistant to such disease in Brazil,a methodology was developed to evaluate aggressiveness in S. sapinea isolates on apple fruits and Pinus taeda. Three environments (greenhouse with different regimes of irrigation), two inoculation methods (with and without injury) and three methods of re-isolation of fungus (PDA medium, moist chamber in plastic bags and moist gerbox) had been tested. Results pointed to significant differences (p < 0,05) on aggressiveness in apple fruit and P. taeda seedlings. The better method to re-isolate was moist chamber in plastic bags and the better environment to incubate inoculated seedlings was greenhouse with two diary aspersion of four minutes. Other result was that injury is necessary for a good inoculation of S. sapinea. The characterization results have presented significant differences in relation to aggressiveness and genetic diversity of the pathogen in all tested hosts.Keywords: Breeding; fungus; inoculation; Pinus.

High-Resolution Melting Analysis: a new molecular approach for the early detection of Diplodia pinea in Austrian pine

The differentiation of Diplodia pinea from closely related species, such as Diplodia scrobiculata and Diplodia seriata, and its detection in plant tissue, represented a critical issue for a long time. Molecular screening tools have recently been developed to address this topic. In this study we applied one of the most sensitive and rapid diagnostic screening method so far developed, called High-Resolution Melting Analysis (HRMA), to detect D. pinea in Austrian pine ( Pinus nigra). HRMA exploits differences in the melting behaviour of PCR products to rapidly identify DNA sequence variants without the need for cumbersome post-PCR methods. We developed a HRMA method to detect specific fungal sequences in the mitochondrial small subunit ribosome gene (mt SSU rDNA). The reliability of this technique was firstly assessed on DNA extracted from pure cultures of D. pinea and closely related species. Amplicon differences were screened by HRMA and the results confirmed by direct DNA sequencing. Subsequently, HRMA was tested on DNA from symptomatic and symptomless pine shoots, and the presence of the fungus was also confirmed by both conventional and molecular quantitative approaches. The HRMA allowed the distinction of D. pinea from closely related species, showing specific melting profiles for the each pathogen. This new molecular technique, here tested in a plant–fungus pathosystem for the first time, was very reliable in both symptomatic and symptomless shoots. HRMA is therefore a highly effective and accurate technique that permits the rapid screening of pathogens in the host.

Distribution of Diplodia pinea and its genotypic diversity within asymptomatic Pinus patula trees

Diplodia pinea (= Sphaeropsis sapinea) is an endophytic fungus and opportunistic canker pathogen of Pinus spp. The diversity of this fungus has been studied at broad geographic scales, but little is known regarding its population structure at smaller spatial scales such as within a single tree. This is despite the importance that diversity in a single tree might hold for understanding the biology of the fungus, especially the role of the endophytic or asymptomatic phase in disease development. Moreover there was not information regarding the distribution of the fungus within healthy trees and its persistence. The genotypic diversity of these isolates was investigated using microsatellite markers. Five polymorphic markers were developed and these were used together with eight previously developed markers and vegetative compatibility tests to study the genotypic diversity of D. pinea isolates. In this study, D. pinea was isolated for the first time in the well structured stems of healthy P. patula trees along with branches and cones. From a total of 44 isolates collected from five trees, 39 microsatellite haplotypes and 32 vegetative compatibility groups (VCG’s) were identified. The results indicate high genotypic diversity of D. pinea within individual asymptomatic trees which will lead to disease outbreak when trees are physiologically stressed.

Can the emergence of pine Diplodia shoot blight in France be explained by changes in pathogen pressure linked to climate change?

Abstract Sphaeropsis shoot blight, caused by Diplodia pinea and Diplodia scrobiculata, damage conifers throughout the world. In France, the first disease outbreaks were reported during the 1990s. The factors associated with the pathogen presence in stands and the relationship between pathogen and disease distributions were analysed in order to understand the Sphaeropsis emergence. Eighty‐two stands of Pinus nigra, Pinus sylvestris, Pinus pinaster and Pinus radiata were visited. Cones were collected on the ground to assess the pathogen frequency. Diplodia spp were isolated and determined by a species‐specific PCR test. The role of potential explaining factors of D. pinea prevalence on cones was analysed by logistic regression. D. pinea was the dominant species in visited stands. The main factors influencing the pathogen presence selected in the models were host species (the pathogen being less frequent on P. pinaster than on P. nigra and P. sylvestris cones), winter temperature and summer rain, which were both positively correlated with cone colonization. The climate became more favourable to D. pinea presence within the last 15 years compared with the previous 30‐year period. By contrast, future climatic changes over the next 40 years should have far less impact on the pathogen presence.

Characterization of a novel dsRNA element in the pine endophytic fungus Diplodia scrobiculata

Diplodia scrobiculata and Diplodia pinea are endophytic fungi associated with dieback and cankers of mainly Pinus spp. in many parts of the world. These two fungi are closely related and have, in the past, been considered to represent two morphological forms (A and B morphotypes) of D. pinea. dsRNA elements are known to occur in both D. scrobiculata and D. pinea. Two dsRNA elements from D. pinea, SsRV1 and SsRV2, have been characterized previously. The aim of this study was to characterize a third dsRNA element that is most commonly associated with D. scrobiculata and to determine its phylogenetic relationship to other mycoviruses. The 5018-bp genome of this element was sequenced, and it is referred to as D. scrobiculata RNA virus 1, or DsRV1. It has two open reading frames (ORFs), one of which codes for a putative polypeptide with a high degree of similarity to proteins of the vacuolar protein-sorting (VPS) machinery, and the other for an RNA-dependent RNA polymerase (RdRp). Phylogenetic comparisons based on amino acid sequence alignments of the RdRp revealed that DsRV1 is closely related to a dsRNA element isolated from Phlebiopsis gigantea (PgV2), and they grouped separately from virus families in which mycoviruses have previously been described. Although D. pinea and D. scrobiculata are closely related, DsRV1 does not share high sequence identity with SsRV1 or SsRV2, and they probably have different recent evolutionary origins.

Interaction Between Diplodia pinea and D. scrobiculata in Red and Jack Pine Seedlings

Sphaeropsis sapinea sensu lato is a conifer fungal pathogen that causes shoot blight and stem cankers. Recently, the former S. sapinea has been divided into two species, Diplodia pinea and D. scrobiculata. The aims of the study were to determine the contribution of each species in disease development on red and jack pines by means of co-inoculations and molecular identifications, and to evaluate how the presence of each species affects the development and aggressiveness of the other. Symptom severity (distance below the inoculation site at which necrotic needles were observed) and identification length (the maximum distance from inoculation site from which either D. pinea or D. scrobiculata was identified using molecular methods) were recorded 4 weeks after inoculating wounded seedlings with agar plugs colonized by these pathogens. The results suggested that D. pinea was much more aggressive on both hosts than D. scrobiculata. When a seedling was co-inoculated with these pathogens, the symptom development appeared to be mainly due to D. pinea. The presence of D. pinea also interfered with the establishment of D. scrobiculata in the plant tissue. However, D. scrobiculata showed antagonism toward D. pinea. When both pathogens co-occurred in a single seedling, symptom severity caused by D. pinea was less than when D. pinea alone was present.

Trichoderma atroviride promotes growth and enhances systemic resistance to Diplodia pinea in radiata pine (Pinus radiata) seedlings

SummaryRoot drench application of Trichoderma atroviride isolates R32, R33, R40 and R84 promoted the growth of potted radiata pine seedlings. After 6 weeks, seedlings treated with R33 and R84 had thicker stems and greater stem and root biomass (p < 0.05) than untreated controls. Treatment with R32 increased seedling root biomass whilst R40 increased stem diameter. None of the isolates affected seedling height. One isolate, R33, induced systemic resistance to stem inoculation with Diplodia pinea and reduced dieback incidence by 20% compared with untreated controls. To our knowledge, this is the first report of systemic induced resistance by Trichoderma in a pine species. Furthermore, seedlings that were treated with R33 (root drench) plus foliar application of methyl jasmonate (MeJA) expressed elevated peroxidase activity in their stems 2 weeks later, compared with seedlings treated only with MeJA. Because R33 itself did not affect peroxidase activity, this may be indicative of treatment synergy or defence potentiation by R33. Curiously, R33 + MeJA induced terpenoids but suppressed phenylalanine ammonia‐lyase activity suggesting possible trade‐offs between phenolic and terpenoid defence pathways in the treated seedlings.

Differential effects of nutrient availability on the secondary metabolism of Austrian pine (Pinus nigra) phloem and resistance to Diplodia pinea

SummaryNutrient availability can modulate the incidence and severity of plant diseases. We examined the effects of fertility on (i) host resistance and (ii) secondary metabolite production in the Pinus nigra Arnold –Diplodia pinea (Desm.) Kickx pathosystem. Following preconditioning of trees to one of three fertility levels for one growing season, phloem tissue of one branch was harvested for chemical analyses while another branch on the same whorl was inoculated with the fungus to assess resistance. Nutrient availability had concave and convex quadratic effects on concentrations of total monoterpenes and total phenolics respectively, but had no effect on fungal lesion lengths. Lesion lengths were negatively correlated with lignin, suggesting that this compound is an important factor in resistance. Counteracting effects of nutrient availability on phenolic and terpenoid pathways may explain why fertility had no effect on expression of disease resistance.

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

SummaryDiplodia 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.

Phylogenic diversity of fungal endophytes in Spanish stands of Pinus halepensis

Fungal endophytes were collected from 168 Pinus halepensis trees sampled in 55 Spanish stands. In total, 229 endophytic isolates were grouped into 92 morphotypes according to their mycelium features. Thus, twigs enclosed 63.76% of the total endophyte isolates and needles 36.24%. Likewise, twig samples also yielded a higher endophyte species richness. Analysis of ITS rDNA region generated 38 different Ascomycota taxa and confirmed the endophytic stage of several pathogens previously associated with the Pinus halepensis decline in Spain. Naemacyclus minor, Brunchorstia pinea, Lophodermium pinastri, Phomopsis sp., Diplodia pinea, Pestalotiopsis besseyi and Truncatella angustata were isolated. Sequencing of LSU rDNA region verified the 38 taxa and contributed to infer their phylogenic relatedness using the Neighbour-Joining method. In the bootstrap consensus tree five classes were observed. Dothideomycetes resulted to be the dominant class because of its high isolation frequency (52.4%) and richest species composition (39.5%). On the contrary, class Eurotiomycetes was the least abundant (3.5%) and along with class Pezizomycetes harboured the lowest species richness (7.9%). Class Sordariomycetes and Leotiomycetes had intermediate abundance and species composition percentages. This study represents the first work concerning the taxonomy of the fungal endophytic community of the Mediterranean host species Pinus halepensis using molecular tools. The data provided here may help to establish the cause of Pinus halepensis decline.

Predicting sapstain and degrade in fallen trees following storm damage in a Pinus radiata forest

Storm damage in production forests constitutes a major source of economic loss world wide, yet the retrieval of salvageable timber remains problematic. In particular, an inability to anticipate when sapstain and degrade will appear hampers the planning of log recovery operations. A study was conducted to monitor the deterioration of fallen trees following two winter storms causing wind and snow damage in a Pinus radiata plantation forest in the upper South Island of New Zealand. Percentage sapstain, incidence of basidiomycete decay fungi, and frequency of bark beetle infestation increased, while percentage sapwood moisture content decreased, over a period of 1 year. These changes proceeded more rapidly in fallen trees that were severed at stump height, to simulate breakage, than in those that were left partially rooted. There was little beetle activity at the time of the storms, but Arhopalus ferus (Coleoptera: Cerambycidae), and Hylastes ater, Hylurgus ligniperda and Pachycotes peregrinus (Coleoptera: Curculionidae: Scolytinae), were collected in flight traps during the following spring and summer. The predominant fungal species associated with sapstain was Diplodia pinea, while Ophiostoma piceae and Grosmannia huntii were isolated near the end of the period. The main decay fungi obtained were Phlebiopsis gigantea, Stereum sanguinolentum, and Schizophyllum commune. A generalized linear mixed model was constructed to predict the development of sapstain in fallen trees for conditions prevailing during the study after a storm at the same time of year. According to the model, a 10m long butt log of 22cm mid length diameter will have minimal stain (<10% of the cross sectional area affected) when cut from severed stems up to 4 months after the storm; if taken from still-rooted trees this period will extend to 1 year. However, because of large between-tree variation, economically productive log recovery will also depend on the proportion of trees that lie below an acceptable sapstain threshold. Further research is needed to determine regional and seasonal influences on the development of sapstain in fallen trees.

Diplodia scrobiculata found in the southern hemisphere

Summary 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.