Botryosphaeriaceae associated with Eucalyptus canker diseases in Colombia

The identities of Botryosphaeriaceae causing cankers on Eucalyptus in Colombia were investigated using morphological and DNA sequence comparisons. The pathogenicity of the species was also assessed on 42 Eucalyptus grandis clones planted at four different sites. Two species of the Botryosphaeriaceae were found to occur on E. grandis in Colombia. Neofusicoccum ribis was the more common species, and also the most pathogenic. Botryosphaeria dothidea was found only in one zone of Colombia, and was also less pathogenic than N. ribis. These two species could be distinguished easily based on DNA sequences of the ITS1/ITS2 rDNA region and EF1‐α, in addition to conidial sizes. Significant differences in resistance of clones to these pathogens were also evident from the various trials.     

Botryosphaeriaceae associated with die‐back of Schizolobium parahyba trees in South Africa and Ecuador

Die‐back of Schizolobium parahyba var. amazonicum is a serious problem in plantations of these trees in Ecuador. Similar symptoms have also been observed on trees of this species in various parts of South Africa. The most common fungi isolated from disease symptoms on S. parahyba var. amazonicum in both locations were species of the Botryosphaeriaceae. The aim of this study was to identify these fungi from both Ecuador and South Africa, and to test their pathogenicity in greenhouse and field trials. Isolates obtained were grouped based on culture morphology and identified using comparisons of DNA sequence data for the internal transcribed spacer (ITS) and translation elongation factor 1α (TEF‐1α) gene regions. The β‐tubulin‐2 (BT2) locus was also sequenced for some isolates where identification was difficult. Three greenhouse trials were conducted in South Africa along with a field trial in Ecuador. Neofusicoccum parvum was obtained from trees in both areas and was the dominant taxon in South Africa. Lasiodiplodia theobromae was the dominant taxon in Ecuador, probably due to the subtropical climate in the area. Isolates of Neofusicoccum vitifusiforme (from South Africa only), Neofusicoccum umdonicola and Lasiodiplodia pseudotheobromae (from Ecuador only) were also obtained. All isolates used in the pathogenicity trials produced lesions on inoculated plants, suggesting that the Botryosphaeriaceae contribute to the die‐back of S. parahyba trees. While the disease is clearly not caused by a single species of the Botryosphaeriaceae in either region, N. parvum has been introduced into at least one of the regions. This species has a broad host range and could have been introduced on other hosts.     

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.     

Neofusicoccum parvum associated with pomegranate branch canker in Iran

Pomegranate (Punica granatum) is native to Iran and neighbouring countries and is negatively affected by a wide range of diseases. Canker and dieback diseases caused by Cytospora punicae and members of the family Botryosphaeriaceae are the main trunk diseases on pomegranate. In summer 2017, progressive dieback of branches on some young pomegranate trees was observed in orchards of the Gorgan Region (Golestan province, Northeast Iran). Fungal colonies with similar colony colour were isolated from symptomatic tissues. The identity of the causal agent was determined as Neofusicoccum parvum, based on a polyphasic taxonomic approach including morphological features of conidiomata and phylogenetic inference based on the ITS‐rDNA region and translation elongation factor 1 alpha (tef1‐α) gene. Koch's postulates were fulfilled using an excised shoot method. This study provides the first report on the occurrence of N. parvum as a cause of branch canker on pomegranate in Iran.     

First report of Lasiodiplodia theobromae and Pseudofusicoccum stromaticum causing dieback in Syzygium malaccense tree in Brazil

Syzygium malaccense is a woody species, known as Malay apple, which is grown as an ornamental or for fruit production in Brazil, mainly in the northern and north‐eastern regions. A high incidence of dieback has been observed in Malay apple in ornamental situations, including streets and gardens. Morphological identification, sequencing analysis of TEF+ITS gene regions and a pathogenicity test confirmed that fungi in the Botryosphaeriaceae cause the disease. This is the first report of Lasiodiplodia theobromae and Pseudofusicoccum stromaticum causing dieback on S. malaccense trees in Brazil.     

河北省葡萄座腔菌科菌物系统分类研究

【目的】葡萄座腔菌科菌物是引起众多经济及生态造林树种枝干溃疡、枝枯类病害的病原生物,在世界各地均有广泛的分布。本研究对河北省内,与经济林木病害相关的葡萄座腔菌科菌物进行系统分类研究,目的在于揭示该科菌物在特定地区的地理分布特征,明确该类菌物所致树木枝干溃疡、枝枯类病害的寄主植物范围,为河北省树木溃疡、枝枯类病害的控制提供理论依据。【方法】在对河北省树木溃疡类病害系统调查的基础上,本研究根据菌物无性型及有性型的形态学特征、rDNA-ITS序列特征对包括河北省石家庄在内的8个市(县、区)25个不同地点的葡萄座腔菌科菌物进行分类鉴定和系统分类。【结果】共获得葡萄座腔菌科菌物5种73株,分别属于Botryosphaeria dothidea、B.stevensii、B.obtusa、B.sarmentorum和Phyllosticta citrichinaensis。其中B.dothidea为河北省葡萄座腔菌科优势菌物种,占全部检出菌物菌株总数的77%。葡萄座腔菌科菌物侵染杨树、柳树、苹果、核桃等28种植物,其中包括大叶黄杨、构树、火炬树、连翘、花椒和刺梅等6种新寄主植物。【结论】B.dothidea不仅危害杨树等传统寄主,而且危害花椒等新寄主植物以及入侵树种火炬树,是危害河北省树木健康生长的最为重要、分布最为广泛的枝干病原菌物,有必要对其溃疡致病性分化和种群遗传分化进行深入分析。来自柿树、花椒等寄主的10个具有Dothiorella型分生孢子的菌株形成单系类群,提示这些菌株可能是与B.sarmentorum和B.iberica不同的一个系统发育学新种。     

Occurrence and pathogenicity of Neofusicoccum parvum and N. mangiferae on ornamental Tibouchina species

Species in the Botryosphaeriaceae are important canker pathogens of woody plants, including Eucalyptus spp. The recent discovery of the Eucalyptus pathogen, Chrysoporthe austroafricana, on ornamental Tibouchina trees raised the question as to whether Tibouchina spp. might be alternative hosts for other Eucalyptus pathogens. Therefore, the aim of this study was to consider whether species of the Botryosphaeriaceae occurring on Eucalyptus spp. might also occur on ornamental Tibouchina spp. Isolations were made from Tibouchina trees in South Africa, New Zealand and Australia. Isolates were identified using morphological characteristics and DNA‐based techniques. Neofusicoccum parvum and N. mangiferae were identified from the samples. Pathogenicity trials on T. urvilleana showed that both species are pathogenic to this host.     

The pathogenic potential of endophytic Botryosphaeriaceous fungi on Terminalia species in Cameroon

In Cameroon, native Terminalia spp. represent an important component of the forestry industry, but limited information is available regarding the fungal pathogens that affect them. The Botryosphaeriaceae are endophytic fungi and latent pathogens that can result in wood stain, cankers, die‐back and death of trees, particularly when trees are under stress. The aim of this study was, therefore, to identify and characterize the Botryosphaeriaceae occurring as endophytes of Terminalia spp. in Cameroon, as part of a larger project to identify potential pathogens of these trees in the country. Samples were collected from three Terminalia spp. in the Central, Southern and Eastern Regions and the resultant Botryosphaeriaceae were identified using morphology and DNA sequence comparisons for the ITS and tef 1‐α gene regions. Furthermore, inoculation trials were conducted to consider the relative pathogenicity of the isolates collected. The majority of isolates (88%) represented species of Lasiodiplodia, including L. pseudotheobromae, L. theobromae and L. parva. The remaining isolates were identified as Endomelanconiopsis endophytica. Pathogenicity trials on young T. mantaly and T. catappa trees revealed that L. pseudotheobromae was the most pathogenic species followed by L. theobromae.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Functional roles of nematodes associated with the walnut twig beetle and eastern black walnut in the inland northwest

Beetles (Scolytinae) form intimate associations with a taxonomically and functionally diverse suite of nematodes that are phytopathogens, fungal feeders, and entomoparasites. Despite their ubiquity, the ecological significance of nematodes in the lifecycles of economically important bark and ambrosia beetle species (Curculionidae: Scolytinae) and associated plant diseases remains largely unexplored. Thousand cankers disease (TCD) is caused by the walnut twig beetle (WTB, Pityophthorus juglandis Blackman) and the fungus Geosmithia morbida (Kolařík, Freeland, Utley & Tisserat; Ascoymycota: Hypocreales) and causes foliar senescence, progressive crown dieback, and mortality in black walnut (Juglans nigra L.) throughout western North America. In this study, nematodes recovered from P. juglandis and J. nigra in Idaho (ID) and Washington (WA) were identified morphologically and by constructing multilocus phylogenies to infer taxonomic relationships to taxa for which molecular data were available. We conducted assays to determine the extent to which nematodes feed and reproduce on G. morbida and other fungi commonly found in galleries of P. juglandis. Inoculation experiments were conducted to determine the effect of nematodes on the area of subdermal necrotic lesions (cankers) caused by G. morbida in branches of mature J. nigra and stems of seedlings. The phoretic nematode Bursaphlenhus juglandis (Ryss, Parker, Alvarez-Ortega, Nadeler & Subbotin) was frequently found under elytra of WTB in all locations, and a free-living nematode (Panagrolaimus sp.) was also widespread and found in the bark of mature trees. Both B. juglandis and Panagrolaimus sp. reduced the size of cankers caused by G. morbida in seedlings and branches of mature trees, respectively. However, these species may play opposite roles as disease synergists and antagonists based on the observation that exudates and/or microbiota associated with Panagrolaimus sp., but not B. juglandis destroyed G. morbida colonies in culture. Furthermore, B. juglandis contributed to foliar symptoms in seedlings inoculated with G. morbida. An entomoparasitic nematode (Aphelenchoididae), most closely resembling an Ektaphelenchus sp., was also found in the haemocoel of WTB. Infection rates were positively related to beetle population sizes as inferred from emergence rates. Ditylenchus sp. was also found in incubated walnut wood in WA and Rhabtidolaimus sp. was phoretic on P. juglandis and found in incubated walnut wood in WA and ID. The community of nematodes in J. nigra in WA and ID differed substantially from what has been observed associated with J. nigra in its native range.     

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.     

Fungi in foliage and shoots of Fraxinus excelsior in eastern Ukraine: a first report on Hymenoscyphus pseudoalbidus

In eastern Ukraine, the first symptoms of dieback on common ash (Fraxinus excelsior) were observed in 2010, as sparse flushing of leaves, bark necrosis and wood discoloration of shoots. The aim of this study was to assess possible causal agents of the damage by studying fungal communities in both symptomatic and healthy‐looking shoots, and leaf petioles. Field sampling was carried out in 2010 in Kharkiv and Sumy regions in eastern Ukraine and included 68 segments of symptomatic shoots, 68 segments of healthy‐looking shoots and 240 segments of petioles. DNA isolation from individual segments and direct sequencing of fungal ITS rRNA resulted in 430 fungal sequences representing 29 distinct taxa. Results showed that Hymenoscyphus pseudoalbidus – the primary causal agent of ash dieback in Europe – was present at low proportion (5.6%) in symptomatic shoots. Other more frequently detected fungi were Epicoccum nigrum, Venturia fraxini, Colletotrichum truncatum, Aureobasidium pullulans, Alternaria alternata, Alternaria sp. and Lophiostoma corticola. In conclusion, the study reports on the first incidence of ash decline in the Ukraine and other possible causal agents of damage which may help to evaluate and forecast the future situation with F. excelsior stands in the region.     

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.     

Status of thousand cankers disease on eastern black walnut in the eastern United States at two locations over 3 years

Thousand cankers disease (TCD), a lethal fungal dieback of eastern black walnut (Juglans nigra), caused by Geosmithia morbida, and spread by the walnut twig beetle, Pityophthorus juglandis, was documented in 2009 to be very destructive in the western United States and was identified in the native range of J. nigra at Knoxville, Tennessee, in 2010, and in 2011 at Richmond, Virginia. Beginning late 2010, we studied branch dieback levels (per cent live crown) and new TCD symptom development at these two quarantined locations monthly for 3 years. Of the 106 trees studied (53 at each location), 31 trees had low live crown ratings of 70 to 0% with little change over the 3 years of the study. One per cent of the trees developed new symptoms on a per‐year basis. Thus, a moderate level of TCD (mean = 76% live crown) was present in these two locations, and most trees were in a quiescent or dormant TCD condition for 3 years, an important finding not previously reported. We found new TCD symptoms developed in Richmond in 2011 and 2012 when precipitation from January 1 to the end of August was low (60–64 cm), and not when the precipitation in Richmond was higher (99 cm). In late 2012, in Richmond, soil water potential assays indicated that some black walnut trees were under severe physiological stress (?15 bars). In contrast, in 2013, high precipitation levels (99 and 130 cm) and high soil water potentials (?0.1 to ?3 bars) at both locations were associated with extensive new foliage and stem growth and recovery from TCD. Further research is needed on water relationships in regard to TCD and black walnut health.     

Diversity and pathogenicity of Botryosphaeriaceae on declining Ostrya carpinifolia in Slovenia and Italy following extreme weather conditions

Unusual and extensive dieback of European hop hornbeam (Ostrya carpinifolia) has been observed in western Slovenia and northern Italy in recent years, when extreme drought and higher temperatures were recorded. A preliminary study identified Botryosphaeria dothidea as a potential causative agent of the dieback. Further characterization of intra- and interspecies diversity of Botryosphaeriaceae collected from O. carpinifolia and other tree species in the affected area was achieved based on anamorph morphology, sequence data from the ITS rDNA and EF1-??, PCR?CRFLP analysis and AFLP profiles. The majority of the isolates analyzed were identified as B. dothidea, and in vitro pathogenicity tests re-confirmed the fungus to be an important cause of the disease. Insight into the B. dothidea population, diversity based on AFLP markers indicates that the dieback observed in O. carpinifolia is probably associated with a heterogenous population of B. dothidea, which emerged from a latent state in response to changed climate conditions. Isolates with Dothiorella-like conidia were also identified during the survey, but these were collected more rarely and appear to represent undescribed species. Isolates from Dothiorella genus expressed low pathogenicity in pathogenicity tests and proved no impact on the pathogenic ability of B. dothidea.     

Canker and dieback on Platanus x acerifolia caused by Diaporthe scabra

Cankers and dieback on London plane caused by Diaporthe scabra have been observed in urban plantings in Catania, Italy. Symptoms were delayed spring flush, trunk cankers, small chlorotic leaves associated with dead terminal or lateral branches and an early defoliation. Isolations from infected wood on PDA led to the isolation of the anamorph Phomopsis scabra with α‐conidia production. The infected wood placed in a moist chamber developed pycnidia with α and β conidia and perithecia of the teleomorph D. scabra. Sequence analysis of the internal transcribed spacers ITS1 and ITS2, the ribosomal intragenic spacer beta‐tubulin 1 and beta‐tubulin 2 genes, congruently indicated that the D. scabra isolate is phylogenetically related to two strains of D. helianthi isolated in Italy from sunflower and to strains of D. eres. Inoculations on young plants of London plane growing in pots confirmed the pathogenicity of the isolated fungus. To our knowledge, this is the first report of the disease in Italy.     

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.     

Cultural characteristics and pathogenicity of Melanconium juglandinum

Cultural characteristics and pathogenicity of Melanconium juglandinum, the agent of the black pustular dieback of Juglans spp. in Europe were investigated. Melanconium juglandinum was consistently isolated from diseased twigs and branches of Persian (English) walnut trees (Juglans regia) in Central Italian Apennine valleys. In growth and conidia germination tests, the fungus was shown to be active within a broad range of temperatures (5–30°C), with the optimum being between 20 and 30°C. Monthly artificial inoculations performed over a period of 2 years with mycelial plugs on 2-year-old seedlings of Persian (English) walnut developed symptoms within 15 days with maximum canker length occurring when inoculation was carried out between June and August. Koch's postulates were fulfilled and M. juglandinum was proved to be a virulent pathogen.     

Pathogenicity to alder of Phytophthora species from riparian ecosystems in western Oregon

Described as one of the most destructive pathogens of agricultural crops and forest trees, Phytophthora is a genus of microorganisms containing over 100 known species. Phytophthora alni has caused collar and root disease in alders throughout Europe, and a subspecies has recently been isolated in North America. Reports of canopy dieback in red alder, Alnus rubra, prompted a survey of their overall health and to determine whether P. alni was present in western Oregon riparian ecosystems. Over 1100 Phytophthora isolates were recovered, representing 20 species and 2 taxa. Phytophthora‐type cankers were observed in many trees, and their incidence was positively correlated with canopy dieback. High levels of mortality for red alder were not observed, which suggests these Phytophthora species are not aggressive pathogens. To test this hypothesis, three stem wound inoculations and one root dip were conducted on red alder seedlings using 13 Phytophthora species recovered from the riparian survey. Ten of the 13 Phytophthora species produced significant lesions in at least one pathogenicity test. Phytophthora siskiyouensis produced the largest lesions on red alder from the two stem wound inoculation tests conducted under summer conditions, while P. taxon Pgchlamydo caused the largest lesions during the winter stem wound inoculation test. Phytophthora gonapodyides, P. taxon Pgchlamydo and P. siskiyouensis have previously been found associated with necrotic alder roots and bole cankers in the field, and with the pathogenicity results reported here, we have established these species as causes of Phytophthora root disease and Phytophthora bole canker of alder in Oregon. While none of the Phytophthora species were especially aggressive towards red alder in the pathogenicity tests, they did cause localized disease symptoms. By weakening the root systems or boles of alders, the Phytophthoras could be leaving alders more susceptible to other insects and pathogens.