First detection of Bursaphelenchus mucronatus (Nematoda: Aphelenchoididae) on Monochamus sutor (Coleoptera: Cerambycidae) in Romania

As a result of the detection of the pinewood nematode Bursaphelenchus xylophilus in Portugal, and its subsequent spread to Spain, intense surveys were conducted to screen for the presence of Bursaphelenchus species in Romania. Herein, we report recent surveys of insects potentially vectoring Bursaphelenchus species collected using trap trees or pheromone‐baited traps placed in the forest. Trap felled spruce trees (Picea abies) and pheromone‐baited traps were installed in six different counties in Romania (Bra?ov, Sibiu, Suceava, Hunedoara, Timi? and Dâmbovi?a). Ten different species of insects distributed among Curculionidae and Cerambycidae were obtained. Nematodes were extracted from insects and observed to validate the presence of Bursaphelenchus specimens. One female identified as Monochamus sutor was the only specimen carrying nematodes in the genus Bursaphelenchus. Nematodes were identified as B. mucronatus based on morphological and molecular features. This is the first detection and report of natural spread of B. mucronatus in Romania. The absence of B. xylophilus was confirmed in the areas of Romania surveyed in this work.     

Discula quercina as a possible causal agent of dieback on oak trees in Iran

During spring and summer of 2016, dieback symptoms including blights of leaves, twigs, and acorns were observed on current year shoots of Quercus infectoria in the Zagros oak forests of Iran. The fungus isolated from damaged tissues was identified as Discula quercina based on both morphological characteristics and ITS sequencing. To fulfil the Koch's postulates, a representative isolate was inoculated onto shoots of Q. infectoria and Quercus libani in both laboratory and forest conditions. Responses of the two oak species to inoculation with D. quercina were examined under laboratory and forest conditions in a completely randomized experiment. Discula quercina was clearly involved in oak dieback, and Q. infectoria was more susceptible than Q. libani to damage by the pathogen. This is the first record of the occurrence and pathogenicity of the fungus Discula quercina on Quercus infectoria. The fungus is considered as an emerging pathogen on oak trees in Zagros forests in Iran. Furthermore, the pathogenicity of the Discula quercina on Q. libani under laboratory and forest conditions increases the potential importance of this pathogen in Zagros forests.     

First report of Ceratocystis platani in Albania

The fungus Ceratocystis platani was detected in various localities of the Gjirokastër prefecture in southern Albania, where it was causing widespread mortality on Platanus orientalis trees. The identification of the fungus was based on both morphological characteristics in culture and DNA sequencing. The pathogenicity of C. platani was confirmed in inoculations on seedlings of P. orientalis. To our knowledge, this is the first report of C. platani in Albania.     

Biscogniauxia nummularia infecting beech (Fagus sylvatica) trees and sympatric plants of the sedge Carex brevicollis

Biscogniauxia nummularia is known for its association with beech (Fagus sylvatica), on which it occurs as a saprotroph and a pathogen causing strip cankers following water stress. This fungus has also been reported as a dominant endophytic species in plants of the sedge Carex brevicollis growing in the understory of beech forests and adjacent grasslands in Sierra de Urbasa (Navarre, Spain). In this area, stromata of B. nummularia were observed in dead and living wood of beech trees at several locations where plants of C. brevicollis also contained the fungus as an endophyte. Pure cultures obtained from stromata of B. nummularia on F. sylvatica trees were compared to endophytic isolates from symptomless C. brevicollis. Culture morphology and micromorphology as well as rDNA sequences of the internal transcribed spacer (ITS) regions were identical, suggesting that B. nummularia from beech can also live endophytically in C. brevicollis. It is unknown whether the endophytic strains of Carex might have a role as an inoculum source for the infection of beech trees, or whether they represent a dead end in the life cycle of the fungus.     

Helminthosporiella stilbacea associated with African oil palm (Elaeis guineensis) in Brazil

African oil palm (Elaeis guineensis Jacq.) is considered the most productive oleaginous crop due to its high oil production per hectare. In April 2015, E. guineensis leaves showing elliptical necrotic spots with yellowish halo were observed in a commercial plantation in Moju, Pará state, Brazil. A synematous fungus was consistently observed associated with the necrotic spots. The aim of this study was to identify this fungus associated with E. guineensis in Brazil. Based on morphology and DNA sequence data of the internal transcribed spacer rDNA, the fungus was identified as Helminthosporiella stilbacea. Helmintosporiella stilbacea has been reported associated with leaf spots on E. guineensis in Africa, Ghana, Sudan and Zambia. To the best of our knowledge, this is the first report of H. stilbacea associated with E. guineensis in Brazil.     

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

Diplodia 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.     

Genetic diversity and population structure of Raffaelea quercus‐mongolicae,a fungus associated with oak mortality in South Korea

Raffaelea quercus‐mongolicae is a fungus associated with oak wilt and deemed to cause extensive oak mortality in South Korea. Since the discovery of this fungus on a dead Mongolian oak (Quercus mongolica) in 2004, the mortality continued to spread southwards in South Korea. Despite continued expansion of the disease and associated significant impacts on forest ecosystems, information is lacking about the origin and genetic diversity of R. quercus‐mongolicae. Restriction‐site‐associated DNA (RAD) sequencing was used to assess genetic diversity and population structure among five populations (provinces) of R. quercus‐mongolicae in South Korea. In total, 179 single nucleotide polymorphisms (SNPs) were identified among 2,639 RAD loci across the nuclear genome of the 54 R. quercus‐mongolicae isolates (0.0012 SNPs per bp), which displayed an overall low expected heterozygosity and no apparent population structure. The low genetic diversity and no apparent population structure among South Korean populations of this ambrosia beetle‐vectored fungus support the hypothesis that this fungus was introduced to South Korea.     

First report of Botrytis leaf blight caused by Botrytis cinerea on Aucuba japonica in China

In May 2019, Aucuba japonica var. variegata, known as an evergreen shrub, was found to have multiple leaf spots in Nanjing City, Jiangsu Province, China. A fungus was isolated from the margins of the lesions and one isolate JS-RL3 was selected for identification and pathogenicity tests. The morphological characteristics of the fungus were similar to Botrytis cinerea. Phylogenetic analysis using concatenated sequences of ITS and G3PDH also showed that isolate JS-RL3 clustered with strains of B. cinerea, a relationship supported by a 100% bootstrap value. This report is the first of B. cinerea causing leaf spots on A. japonica var. variegata.     

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

Leptoglossus occidentalis, an insect native to North America, was inadvertently introduced into Italy about 1999. The insect damages the cones of conifer trees, especially Pinus pinea (Italian Stone pine). Pinus pinea is also affected by Diplodia pinea, a fungus native to Italy, which is becoming an increasing threat because pine trees are becoming more susceptible to it as a consequence of global warming. Because the insect and the fungus both have the pine cones as a common habitat, a possible interaction between them has been postulated. The aim of this study was to ascertain whether L. occidentalis and D. pinea interact on P. pinea cones. The interaction was studied using real‐time PCR on a group of naturally infected insects collected from a forest, and a group raised in the laboratory and artificially inoculated with D. pinea conidia. Molecular analysis showed that D. pinea DNA occurred on both naturally infected and inoculated insects, but with significant differences between the two groups. The rapid and sensitive molecular technique made it possible to detect D. pinea DNA on the bodies of the insects, and to show that the native D. pinea occurred on the exotic insect.     

First report of Rhytisma filamentosum causing tar‐spot disease on Salix koriyanagi

The occurrence of previously unknown tar‐spot disease on Salix koriyanagi was observed during surveys for forest disease outbreaks in Korea. The fungus associated with tar‐spot disease was identified as Rhytisma filamentosum based on morphological characteristics of the fungus and molecular sequencing of the ITS and 28S rDNA regions of two samples. To our knowledge, this is the first report of R. filamentosum on S. koriyanagi from Korea, following its first discovery on Salix integra in Japan.     

Rapid detection of Fusarium circinatum propagules on trapped pine beetles

Pitch canker is a destructive disease of pine caused by the fungus Fusarium circinatum. This taxon is listed as a quarantine fungus for several regional plant protection organizations throughout the world. Whereas long‐distance spread of the disease is made possible through the trade of infected pine seeds, local spread is caused by aerial dispersion or insect transportation of the fungal conidia. Developing a reliable and efficient tool to detect of F. circinatum in insects would be very useful to monitor the local spread of the pathogen. This tool would also provide the means to assess the range of insect species that could serve as potential vector of the fungus. A DNA extraction protocol was optimized and combined with a real‐time PCR test to detect F. circinatum on pine beetles. Using artificially contaminated Ips sexdentatus, it was shown that the test was able to detect down to 10 F. circinatum conidia per individual, and 20 conidia per batch of 10 insects, which is below the lowest inoculum load occurring in nature. With this technique, several batches of up to 10 insects may be analysed simultaneously, with a timescale for analysis reduced to <5 h and without the need for expertise in Fusarium taxonomy. This tool may be useful to monitor potential spread of the pathogen across regions. Using this method, to date, despite F. circinatum foci occurred in Northern Spanish regions across the border in France, the pathogen was not found on I. sexdentatus.     

Hymenoscyphus albidus is not associated with an anamorphic stage and displays slower growth than Hymenoscyphus pseudoalbidus on agar media

Examination of isolates of Hymenoscyphus albidus from France revealed that this fungus does not form an anamorphic stage in culture. The lack of an asexual stage in this fungus is a conspicuous morphological difference to the ash dieback pathogen Hymenoscyphus pseudoalbidus, which is associated with its Chalara fraxinea anamorphic state. In growth studies on malt extract agar (MEA) and MEA amended with ash leaflets (ash leaf malt extract agar, AMEA) at 20°C, isolates of H. albidus grew slower than those of H. pseudoalbidus. On AMEA, the growth of cultures of both species was greatly enhanced.     

First report of Ceratocystis huliohia causing mortality of Metrosideros polymorpha trees on the Island of Kauaʻi,Hawaiʻi USA

The fungus Ceratocystis huliohia was detected for the first time in dead and dying Metrosideros polymorpha trees on the Island of Kaua?i, Hawai?i, U.S.A. Fungal isolates were recovered from diseased trees into culture, and identities were confirmed by diagnostic qPCR assay and DNA sequencing. Growth chamber inoculations confirmed C. huliohia pathogenicity on M. polymorpha seedlings and subsequently the same fungus was recovered, completing Koch's postulates. DNA sequence analysis revealed a unique ITS haplotype for Kaua?i isolates of C. huliohia that has not been found on Hawai?i Island.     

Platypus cylindrus,a vector of Ceratocystis platani in Platanus orientalis stands in Greece

The ambrosia beetle Platypus cylindrus was very common in stands of Platanus orientalis trees in Greece, infesting trees already infected by Ceratocystis platani. The fungus C. platani was isolated from 59% of adult beetles of P. cylindrus collected on the wing in a heavily infected stand of P. orientalis. Transmission of C. platani was demonstrated in 80% of P. orientalis seedlings challenged with P. cylindrus beetles, which were previously fed in cultures of the fungus. In most of the cases, the beetles had bored tunnels in stems, where abundant perithecia of C. platani were observed. Platypus cylindrus adults regularly visited artificially wounded P. orientalis trees in a natural stand. Although this ambrosia beetle normally infests stressed or dead trees, it appears to play a role as a vector of C. platani, especially by visiting wounded trees.     

Xyleborus glabratus attacks and systemic colonization by Raffaelea lauricola associated with dieback of Cinnamomum camphora in the southeastern United States

Laurel wilt, caused by Raffaelea lauricola, is responsible for extensive mortality of redbay and other American members of the Lauraceae in the southeastern United States. Raffaelea lauricola is a mycangial symbiont of the redbay ambrosia beetle (Xyleborus glabratus), and the beetle and fungus were accidentally introduced from Asia. Branch dieback of camphortree (Cinnamomum camphora), an Asian member of the Lauraceae, has been occasionally observed in areas where laurel wilt has decimated redbay populations, and R. lauricola was isolated from such camphortrees. However, the role of X. glabratus and R. lauricola in this branch dieback remains unclear. Examination of camphortrees on Jekyll Island, Georgia showed that healthy‐appearing trees and those with branch dieback had been attacked by X. glabratus, but the trees with branch dieback had four times as many beetle attacks. Raffaelea lauricola was routinely isolated from discoloured xylem near beetle tunnels in healthy trees and those with dieback. Single‐point inoculations with R. lauricola on stems of mature, healthy camphortree trees failed to induce wilt‐like symptoms or branch dieback, although areas of discoloration were scattered throughout the xylem, and R. lauricola was reisolated irregularly at various heights in some inoculated trees. In growth chamber experiments, single‐point inoculations with R. lauricola resulted in systemic colonization but no wilt symptoms or branch dieback in camphortree saplings. In contrast, inoculations at multiple points along the stem (simulating multiple attacks by the vector) caused branch dieback and wilt‐like symptoms, including a brownish, diffuse discoloration of the xylem. Camphortree appears to be more resistant than American species of Lauraceae to the vascular wilt caused by R. lauricola. The fungus does colonize camphortrees systemically, however, and can apparently cause branch dieback. This suggests that the fungus may provide brood material for X. glabratus in Asia as it does in the southeastern United States.     

Ceratocystis smalleyi colonization of bitternut hickory and host responses in the xylem

Colonization of Carya cordiformis sapwood by Ceratocystis smalleyi and subsequent host defence responses following artificial inoculation were investigated using anatomical and histological techniques. Hyphae of C. smalleyi were observed in all sapwood xylem features confirming the ability of the pathogen to invade and colonize the xylem tissues of the host species. The fungus was isolated from within and at the margins of discoloured sapwood areas at 2 and 12 months after inoculation. General host defence responses that included vessel occlusion with gels or tyloses, lipid accumulation, and production of phenolic compounds were observed in xylem tissues of inoculated C. cordiformis stems. Pectic substances, lipids, and to a rare extent, phenolic compounds were detected in vascular gels. The lipid‐rich barriers observed likely prevent lateral expansion of the fungus in the sapwood. Furthermore, lack of fungus sporulation within vessels may restrict axial spread of the fungus. C. smalleyi appears to be a limited vascular wilt pathogen of bitternut hickory based on these observations and previously reported sap flow reduction correlated with multiple infections in artificially inoculated trees.     

First report of Teratosphaeria gauchensis causing stem canker of Eucalyptus in Kenya

Teratosphaeria stem canker is an important disease of Eucalyptus species in many parts of the world where these trees are intensively propagated in plantations. Symptoms similar to those of Teratosphaeria stem canker were observed on Eucalyptus grandis and a E. grandis × E. camaldulensis hybrid clone in the Central Highlands of Kenya. Symptomatic bark samples were collected from two sites and the associated fungus isolated and identified using DNA sequence analyses of multiple gene regions. The pathogen was identified as Teratosphaeria gauchensis. This represents the first report of the disease and the pathogen in Kenya.     

Genotype distribution of Raffaelea quercivora in the oak galleries and its composition in the mycangia of Platypus quercivorus

Raffaelea quercivora is the pathogenic fungus that causes Japanese oak wilt. The female monogynous ambrosia beetle, Platypus quercivorus, carries this fungus in mycangia on the pronotum. These beetles bore galleries in oak trees with their partners to produce offspring, and they deposit fungus on the gallery walls from their mycangia. The offspring mature in the gallery, before loading the fungal pathogen and flying from the gallery to other healthy trees. To investigate the unloading and loading modes of the fungus within the gallery, we developed four polymorphic microsatellite markers for R. quercivora and identified the fungal genotypes in the galleries and mycangia of the beetles. Small wood chips were sampled at 5–10‐mm intervals from the walls of five galleries in a dead Quercus serrata tree. The pronota were also sampled from five female adult beetles. The genotypes of the R. quercivora isolates from the wood chips and pronota were identified using the microsatellite makers. The genotypic analysis showed that each gallery was inhabited patchily by 5–10 genotypes of R. quercivora, and the mycangia of each beetle contained 3–6 genotypes. These results indicate that diverse R. quercivora genotypes are unloaded repeatedly from the mycangia of female beetles onto the gallery wall, which results in their patchy distribution on the walls. When the offspring leave the host tree, the fungal clones that proliferate in the walls are also loaded repeatedly into the mycangia of the mature beetles.     

Dothistroma septosporum on firs (Abies spp.) in the northern Baltics

In the spring of 2008, Dothistroma septosporum, a damaging quarantine pathogen of pine foliage, was documented in Järvselja (southeastern Estonia), apparently for the first time, on white fir (Abies concolor). Thus, a new host species and genus for the fungus were recorded. Infection of white fir by D. septosporum, and also of some other fir species, was monitored annually on a transect proceeding from southeastern Estonia to northern Latvia. As a result, D. septosporum was detected also on silver fir (Abies alba). It is not clear why this fungus with a worldwide distribution appeared on this new host genus definitely in northern Europe.