Diplodia Species Research Articles

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

AbstractDecline 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.

In vitro evaluation of antagonistic potentiality of Trichoderma harzianum against Diplodia spp. phytopathogenics fungi

BackgroundTrichoderma species are able to cause significant changes in the metabolism of host plants, by that means it trigger the plant growth and increasing plant defense to diverse fungal diseases. Trichoderma harzianum has been identified as potential biocontrol agent of many phytopathogenic fungi. Therefore, the aim of this study was to assess in vitro antagonistic activity against three Diplodia isolates utilizing direct and remote confrontations methods.ResultsThe results revealed that T. harzianum inhibited mycelial growth of the three phytopathogenic fungi compared to the untreated control. The percentage of growth inhibition of T. harzianum against Diplodia isolates ranged between 58 and 79% for direct confrontation and between 31 and 46% remote confrontation. Considerable antagonistic abilities of T. harzianum were exhibited against all tested Diplodia isolates. The results of both confrontations showed that the radial growth of the fungal pathogens was statistically significant (P < 0.001) and influenced by the antagonist on 6 days following incubation.ConclusionThe findings may reveal a valuable knowledge that may be further used to find a suitable biological control contrariwise Botryosphaeria dieback caused by Diplodia species.

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

AbstractThe 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.

Pest categorisation of Diplodia bulgarica.

The EFSA Plant Health Panel performed a pest categorisation of Diplodia bulgarica, a clearly defined plant pathogenic fungus of the family Botryosphaeriaceae. The pathogen affects Malus domestica, M.sylvestris and Pyrus communis causing various symptoms such as canker, twig blight, gummosis, pre- and post-harvest fruit rot, dieback and tree decline. The pathogen is present in Asia (India, Iran, Türkiye) and in non-EU Europe (Serbia). Concerning the EU, the pathogen is present in Bulgaria and widespread in Germany. There is a key uncertainty on the geographical distribution of D. bulgarica worldwide and in the EU, because in the past, when molecular tools were not available, the pathogen might have been misidentified as other Diplodia species (e.g. D. intermedia, D. malorum, D. mutila, D. seriata) or other members of the Botryosphaeriaceae family affecting apple and pear based only on morphology and pathogenicity tests. Diplodia bulgarica is not included in Commission Implementing Regulation (EU) 2019/2072. Plants for planting, other than seeds, fresh fruits, and bark and wood of host plants as well as soil and other plant-growing media carrying plant debris are the main pathways for the further entry of the pathogen into the EU. Host availability and climate suitability factors are favourable for the further establishment of the pathogen in the EU. In the areas of its present distribution, including Germany, the pathogen has a direct impact on cultivated hosts. Phytosanitary measures are available to prevent the further introduction and spread of the pathogen into the EU. Diplodia bulgarica satisfies the criteria that are within the remit of EFSA to assess for this species to be regarded as potential Union quarantine pest.

Diaporthe and Diplodia Species Associated with Walnut (Juglans regia L.) in Hungarian Orchards

Walnut (Juglans regia L.) production is a developing sector in Hungarian horticulture, where preharvest fruit rot increasingly causes economic losses. Previously, fungi from the Diaporthaceae and Botryosphaeriaceae families were isolated with a high incidence from rotten fruits. Here, we identify these pathogens from different plant parts (twigs, buds, and shoots) in orchards in the north-east of Hungary, and test their pathogenicity on immature nuts. Diaporthe eres Nitschke and Diplodia seriata De Notaris were identified in isolates from different symptomatic and asymptomatic plant parts based on their ITS (nuclear ribosomal internal transcribed spacer) and tef1 (translation elongation factor EF-1-alpha gene) DNA sequences. Purified monocultures caused rot of immature nuts following in vitro inoculation. Our results suggest that D. eres Nitschke and D. seriata De Notaris lingering on buds and overwintering woody parts may affect the seasonal nuts through wound infection. Infection by Diaporthe and Botryosphaeriaceae species present on woody plant parts, as well as on/in buds has been reported in Mediterranean countries. This is the first report of such stepwise aetiology from a region with continental weather. Climate change, associated weather patterns and the limitations of fungicide use in the European Union, among other factors, could be responsible for the increasing number of infections and economic damage caused by these pathogens.

Isolation, molecular characterization and first report of Dothiorella gregaria associated with fruit rot of walnuts of Jammu and Kashmir, India.

Walnuts are known for their high levels of antioxidants, which are linked to various health benefits. However, challenges related to distribution and storage, as well as the risk of fungal infections, can affect the quality of walnut kernels. Fungal pathogens from the Botryosphaeriaceae family, including Dothiorella species and Diplodia species, can damage fruit and reduce its antioxidant content. To comprehend the cause of fruit rot in walnuts, Dothiorella gregaria isolates were studied using polyphasic methods, including multiple gene sequences and morphological identification, as well as analysis of polyphenol content and pathogenicity. The walnuts kernels purchased from market places of Jammu and Kashmir (J&K), India were observed to be affected by Dothiorella gregaria species causing the quality detoriation and decrease in polyphenol content thus undeniably with decreased antioxidant properties. D. gregaria Infected walnut kernels were having some brown and black spots and some were having white mycelial growth and however, most samples were asymptomatic. Pathogenicity testing revealed that the pathogen was able to develop all the symptoms under experimental conditions and the reisolated pathogen was morphologically similar to D. gregaria. The samples infected with this pathogen showed considerable decrease in polyphenol content, 10.9±2.66 mgGAE/g (mean±standard deviation) thus decreased antioxidant quality as compared to the samples which showed zero incidence of this pathogen, 52.50±4.27 mgGAE/g (mean±standard deviation). Furthermore, the pathogen was studied using polyphasic approach involving morphological, molecular and phylogenetic analysis. Combined nucleotide dataset of nuclear ribosomal ITS and tef1-α revealed that Dothiorella gregaria (NY6) formed a clade with Dothiorlla iberica (MAEC33), Dothiorella sarmentorium (MAEC28) and Dothiorella iberica (CAA905) strains with 83% bootstrap support. Besides, we observed six nucleotide changes, four were insertions or deletions and two were substitutions in the 502-bp region of the ITS rRNA gene when we compared our isolate to the most equivalent sequences submitted to NCBI GenBank. This is the first report of Dothiorella gregaria affecting walnuts purchased from various markets in J&K, India, causing fruit rot in walnuts after harvest. Given that local farmers store and export walnuts, it could pose an emerging threat to their livelihood. Thus, creating post-harvesting interventions for D. gregaria and knowing more about the fruit rot in walnuts can be benefited from morphological and molecular identification using several gene loci, genetic variability in the ITS rRNA gene, and total phenol analysis.

Comparison of Diplodia Tip Blight Pathogens in Spanish and North American Pine Ecosystems.

Diplodia tip blight is the most ubiquitous and abundant disease in Spanish Pinus radiata plantations. The economic losses in forest stands can be very severe because of its abundance in cones and seeds together with the low genetic diversity of the host. Pinus resinosa is not genetically diverse in North America either, and Diplodia shoot blight is a common disease. Disease control may require management designs to be adapted for each region. The genetic diversity of the pathogen could be an indicator of its virulence and spreading capacity. Our objective was to understand the diversity of Diplodia spp. in Spanish plantations and to compare it with the structure of American populations to collaborate in future management guidelines. Genotypic diversity was investigated using microsatellite markers. Eight loci (SS9–SS16) were polymorphic for the 322 isolates genotyped. The results indicate that Diplodia sapinea is the most frequent Diplodia species present in plantations of the north of Spain and has high genetic diversity. The higher genetic diversity recorded in Spain in comparison to previous studies could be influenced by the intensity of the sampling and the evidence about the remarkable influence of the sample type.

Diplodia Species Causing Dieback on Pinus Pinea: Relationship Between Disease Incidence, Dendrometric and Ecological Parameters

ABSTRACT Forest decline is a widespread phenomenon on coasts throughout the Mediterranean basin. Pathogenic fungi are amongst the main causes of forest tree diseases. Diplodia species, having a cosmopolitan distribution, are well-known as pathogens of woody plant hosts including Pinus spp. In recent years, symptoms of shoot dieback, necrosis, twig blight, canopy transparency and trunk cankers have been observed on Pinus pinea trees in Tunisian forests. Nevertheless, this has been less well-studied in North Africa and especially in Tunisia. The purpose of this study is to report the occurrence of Diplodia species causing dieback on P. pinea in two forests in northeastern and the northern Tunisia. A collection of fifty-eight isolates were obtained from symptomatic branches of P. pinea trees. Three Diplodia species were identified: D. africana, D. pseudoseriata and D. scrobiculata by means of morphological characteristics, and phylogenetic analysis. Their incidence on P. pinea trees appeared to be significantly correlated to the dendrometric parameters and ecological factors. A pathogenicity test was conducted on 3-years-old P. pinea seedlings, confirmed the virulence of three Diplodia species. Based on the length of vascular necrosis in the wood of the P. pinea inoculated stem, D. scrobiculata was the most virulent species. Data from the present study provide the first report of the species of Diplodia associated with P. pinea dieback in Tunisia.

Diplodia parva sp. nov., a novel species of the family Botryosphaeriaceae isolated from soil in Korea

A fungal strain designated KNU16-007, belonging to the family Botryosphaeriaceae, was isolated from soil in Daegu, Korea. Phylogenetic analyses based on the concatenated nucleotide sequences of the ITS and TEF-1α regions, showed that the isolate resides in a clade together with Diplodia species but occupies a distinct phylogenetic position. Conidial dimensions of strain KNU16-007 (22.7–29.3 μm × 8.9–10.9 μm) or its conidial length:width ratio were significantly differed from those of the closely related D. sapinea, D. intermedia, D. scrobiculata, D. seriata, D. crataegicola, D. rosacearum, and D. citricarpa clearly indicating morphological differences from these species. Detailed descriptions, illustrations, and discussions regarding the morphological and phylogenetic analyses of the closely related species are provided to support the novelty of the isolated species. The results of phylogenetic analysis and morphological observations indicate that strain KNU16-007 represents a novel species in the genus Diplodia, for which the name Diplodia parva sp. nov. is proposed.

Sphaeropsidin A: A Pimarane Diterpene with Interesting Biological Activities and Promising Practical Applications.

Sphaeropsidin A (SphA) is a tetracyclic pimarane diterpene, first isolated as the main phytotoxin produced by Diplodia cupressi the causal agent of a severe canker disease of Italian cypress (Cupressus sempervirens L.). It was also produced, together with several analogues, by different pathogenic Diplodia species and other fungi and showed a broad array of biological activities suggesting its promising application in agriculture and medicine. The anticancer activity of SphA is very potent and cell specific. Recent studies have revealed its unique mode of action. This minireview reports the structures of SphA and its family of natural analogues, their biosynthetic origins, their fungal sources, and biological activities. The preparation of various SphA derivatives is also described as well as the results of structure‐activity relationship (SAR) studies and on their potential practical applications.

MOLECULAR IDENTIFICATION OF Diplodia seriata De Not. CAUSING DIEBACK EFFECT ON GRAPEVINES AND EVALUATION OF in vitro EFFICACY OF FIVE DIFFERENT SYNTHETIC FUNGICIDES AGAINST THIS DISEASE

The aim of this study was to realize the molecular identification of Diplodia seriata De Not., a member of the Botryosphaericea family, isolated from 2-10 years old vines in vineyards showing symptoms of dieback disease. The susceptibility of the pathogen against the fungicides with the fosetyl-Al+triadimenol, azoxystorbin+difecanozole, fludioxanil+cyprodinil, metrafenone, fluopyram+tebuconazole combinations were evaluated. The isolates obtained from the root and crown parts of the vine samples were identified as D. seriata according to the morphological and molecular methods. In molecular identification, the ITS (Internal Transcribed Spacer) and TUB2 (β-tubulin) gene regions of the isolates were amplified by Real-Time PCR and the nucleotide sequences were obtained in these gene regions. After using the MEGA 7 software, ITS and TUB2 sequences were aligned and a combined phylogenetic tree was made. It has been molecularly confirmed that the D. seriata isolate has a 100% similarity index with Diplodia species according to the phylogenetic analyses. The mean effective concentration (EC50) values of fungicides used with different concentrations (0, 1, 3, 10, 30, 50, 100 μL mL-1) were determined by Probit analysis. Cyprodinil + fludioxonil showed the highest efficacy (100%) at a concentration of 1 μL mL-1. According to EC50 values, cyprodinil + fludioxonil (0.001 μL mL-1) was recorded as the most effective fungicide followed by fluopyram + tebuconazole (0.520 μL mL-1) and, azoxystrobin + difenoconazole (2.958 μL mL-1), respectively.

Morphological and Phylogenetic Resolution of Diplodia corticola and D. quercivora, Emerging Canker Pathogens of Oak (Quercus spp.), in the United States.

In Mediterranean Europe and the United States, oak species (Quercus spp.) have been in various states of decline for the past several decades. Several insect pests and pathogens contribute to this decline to varying degrees, including Phytophthora cinnamomi, Armillaria spp., various insect defoliators, and, in the United States, the oak wilt pathogen Bretziella fagacearum. More recently, two emerging canker pathogens, Diplodia corticola and D. quercivora, have been implicated in causing dieback and mortality of oak species in Europe and in several regions in the United States. In 2019, a fungal survey was conducted in the Mid-Atlantic region of the eastern United States, including Maryland, Pennsylvania, Virginia, and West Virginia, to determine the range and impact of D. corticola and D. quercivora on forest health in the United States. A total of 563 oak trees between red and white oak family members were evaluated across 33 forests spanning 18 counties. A total of 32 Diplodia isolates encompassing three Diplodia spp. were recovered from 5,335 total plugs collected from the 13 of 18 sampled counties. Recovered Diplodia species included D. corticola, D. quercivora, and D. sapinea, as well as Botryosphaeria dothidea, a closely related canker pathogen in the Botryosphaeriaceae. Both D. corticola and D. sapinea were recovered from red and white oak family members, whereas D. quercivora was exclusive to white oak family members and B. dothidea to red oak family members. Of these species, D. corticola was most frequently isolated, followed by D. quercivora, D. sapinea, and B. dothidea. Overall, mortality was low across all sampled counties, indicating that these fungi, at the levels that were detected, are not widely inciting oak decline across the region, but probably are acting opportunistically when the environment is conducive to disease. To better understand the relationships between D. corticola and potentially their geographic origins, a multigene phylogenetic study and corresponding morphological study were conducted. A total of 49 Diplodia isolates from Spain, France, Italy, and the United States were assessed. Across all isolates and geographic regions, D. corticola formed a strongly supported monophyletic clade sister to D. quercivora and included two strongly supported subclades, one that included isolates from Spain and California and a second that included isolates from Italy, Maryland, and West Virginia. Both subclades also exhibited overlapping spore measurements. These results support D. corticola as a cosmopolitan pathogen, native to both Europe and the United States, with the possibility of secondary introductions.

Morphological and molecular characterization of Diplodia spp. associated with apple canker disease in India

In recent years, the incidence of apple canker disease is increasing and assuming an epidemic in major apple growing parts of Jammu and Kashmir. During 2017–18, canker disease incidence was observed in all the apple growing regions surveyed, with characteristic symptoms of smoky canker. Total 40 samples were collected from regions of north, central and south Kashmir from three different varieties, Red Delicious, Royal Delicious and Golden Delicious. Among 40 samples collected total 20 isolates representing all the three varieties and regions were maintained for morpho-molecular characterization. Based on cultural and morphological characteristics, the fungus was identified as genus Diplodia with two morphological groups. Sequence based analysis of the internal transcribed spacer region (ITS) and elongation factor (EF1) gene confirmed the species as Diplodia bulgarica and Diplodia seriata. The Diplodia bulgarica was found predominant species in all the three regions surveyed. The phylogenetic analysis of both the genes clustered the two species in two different groups. The pathogenicity was confirmed on two-year-old potted plants of apple cv. Red Delicious. This study would help in better understanding the role of Diplodia species on symptom development, epidemiology and formulation of improved disease management strategies against apple canker.

Identification and pathogenicity of Diplodia seriata and Diplodia africana related to lentisk dieback in Tunisia

Lentisk dieback has been commonly observed in Tunisian forests. Nevertheless, the identity of the parasitic factors involved in this phenomenon has not yet been reported. Thus, in order to characterize the dieback-associated agents, symptomatic branches were collected from Pistacia lentiscus in the Rimel forest (Northern Tunisia). Two Botryosphaeriaceae species, Diplodia seriata and D. africana, were isolated and identified firstly based on morphological characteristics. Then, molecular identification was performed by sequencing the internal transcribed spacer region (ITS1-5.8S-ITS4), a part of the translation elongation factor 1-α (EF1-α) gene and a partial sequence of the β-tubulin (TUB) gene. The two species were morphologically and phylogenetically distinct from all Diplodia species and represent newly recorded fungal pathogens of lentisk in Tunisia. Pathogenicity tests carried out on asymptomatic detached branches confirmed the virulence of the two species. These findings may confirm D. seriata and D. africana as fungal pathogens associated with lentisk dieback in Tunisia.

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

SummaryIn 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.

Conidial production by &lt;i&gt;Botryosphaeriaceae&lt;/i&gt; species from grapevine shoot lesions in Marlborough vineyards

Botryosphaeriaceae species are important pathogens of grapevines so the effect of environmental conditions on sporulation from naturally infected Sauvignon blanc grapevines in two Marlborough vineyards was studied. Shoot lesions characteristic of Botryosphaeriaceae infection were marked on 24 grapevines in each vineyard for observation between September 2014 and April 2015. Pycnidia on the lesions oozed conidia during or soon after rainfall on five occasions at maximum air temperatures of 8.4—19.9˚C and relative humidities of 77—94.7%. Microscopic observation of the ooze showed conidia characteristic of either Neofusicoccum or Diplodia species in 67% and 50% of samples from Vineyard A and B, respectively. Sixty- nine colonies characteristic of Botryosphaeriaeae species were recovered after plating of conidial ooze and identified by DNA analysis as Diplodia mutila (39.1%), Neofusicoccum australe (30.4%), N. parvum (14.5%), N. luteum (5.8%), D. seriata (7.2%) and N. ribis (3.0%). All species have been reported previously as grapevine pathogens in New Zealand.

Detection of the Diplodia shoot blight and canker pathogens from red and jack pine seeds using cultural methods

The shoot blight and canker pathogens Diplodia pinea and D. scrobiculata commonly and abundantly sporulate on seed cones of red pine (Pinus resinosa) and jack pine (P. banksiana) collected from Wisconsin and Minnesota forests. Cultural methods were used to investigate the incidence of these fungi in seed lots obtained from government nurseries in these states. In each of three replicate trials, seeds of each lot were assigned to four treatments before incubation on semi-selective medium: (1) not surface-disinfested; (2) surface-disinfested; (3) surface-disinfested, then inoculated with D. pinea conidia; or (4) not surface-disinfested, then inoculated with D. pinea conidia. For red pine seeds, the mean percentage positive was 2.7% for treatment 1 and 1.3% for treatment 2. Jack pine seeds were less frequently positive than red pine seeds for both treatments 1 and 2. The Diplodia species cultured was identified as D. pinea in almost every case. Diplodia pinea was much less frequently recovered from seeds that were not surface-disinfested and then inoculated (treatment 4), when compared with seeds that were inoculated with D. pinea after surface-disinfestation (treatment 3). Results confirm the potential for dissemination of D. pinea on red pine and jack pine seeds, and caution is warranted before concluding absence of the pathogen based on results using cultural methods with relatively small numbers of seeds. Although the frequency of pathogen-positive seeds was low, the large numbers of seeds planted in nurseries suggest that seeds may be a potentially important route of entry of D. pinea into nursery beds.

Diplodia sapinea: the main fungal species involved in the colonization of pine shoots in Italy

SummaryDamage to various forest tree species caused by Diplodia sapinea has been reported in Italy since the early 20th century. However, until recently, systematic characterization of this microorganism, based on DNA sequence data, has been limited with only a general description of the fungus in the majority of the reports. To address this and provide a more accurate identification of Diplodia species associated on pine in Italy, we examined a total of 88 Botryosphaeriaceae isolates obtained from symptomatic and asymptomatic material from different hosts located in different geographical regions of Italy. Using molecular analysis, we were able to identify 67 isolates of D. sapinea and eight isolates of D. seriata. Diplodia sapinea was dominant on P. nigra shoots and was also detected on P. halepensis, P. pinea, P. pinaster, P. radiata and P. sylvestris, while D. seriata and other Botryosphaeriaceae appeared to be only occasionally present on shoots. The remaining 13 isolates represented nine different Botryosphaeriaceae species, occurring at low levels. Analysis of molecular variance (amova) based on DAMD‐PCR profiles detected low significant differences among D. sapinea isolates from the different sampling areas throughout Italy.

The complex of Diplodia species associated with Fraxinus and some other woody hosts in Italy and Portugal

Studies on the taxonomy and phylogeny of Diplodia have been hampered by the lack of an ex-type culture linked to the holotype of D. mutila, which is the type of the genus. In this study a large collection of Diplodia strains, obtained from ash and other woody hosts showing V-shaped cankers and branch dieback, were identified based on morphological characters and DNA sequence data from ITS and EF1-α loci. Results of combined morphological and phylogenetic analyses showed that the Fraxinus isolates from Italy, the Netherlands, Portugal and Spain belong to three distinct species namely Diplodia fraxini, Diplodia mutila and Diplodia subglobosa sp. nov. An epitype was designated for Diplodia mutila, with associated ex-epitype cultures. The name D. fraxini is re-instated and a neotype designated. Two species, Diplodia seriata and Diplodia pseudoseriata were reported for the first time on Fraxinus spp.

Virulence affected by assay parameters during grapevine pathogenicity studies with Botryosphaeriaceae nursery isolates

This study assessed the symptoms that developed when 114 Botryosphaeriaceae isolates from grapevine nursery plant materials were used to inoculate excised green shoots and 1‐year‐old rooted canes of Sauvignon blanc. The experiments showed that all isolates and species were able to produce lesions. Overall, the Neofusicoccum species were shown to be highly pathogenic in both tissue types while the Diplodia species were highly pathogenic on canes but not on green shoots. Isolates of the most prevalent species, N. luteum and N. parvum, showed varying lesion lengths on excised green shoots and canes. An evaluation of the factors associated with lengths of lesions showed that they were significantly affected by experimental batch which reflected inherent host and environmental factors over time. Reisolation from inoculated canes also indicated that most isolates of all species except D. seriata were able to spread internally beyond the lesions. Genetic variability analysis using UP‐PCR showed that N. luteum isolates were genetically diverse but no association was observed between the phylogenetic group and degree of pathogenicity caused by the isolates. This study demonstrated that all Botryosphaeriaceae species from grapevine nurseries were pathogenic to grapevines and that the lesion lengths varied between species, among isolates within a species and among nursery sources, and was affected by the test method.