Fusarium species in declining wild apple forests on the northern slope of the Tian Shan Mountains in north‐western China

Wild apple forests in the Tian Shan Mountains in north‐western China have been adversely affected by an unknown disease in recent years. Symptoms attributed to this disease that affects wild apple trees include xylem browning and dieback which are suggestive of infection by Fusarium species. Therefore, the research team conducted the first survey for Fusarium in the afflicted wild apple forests. Twig samples with symptoms of xylem browning and dieback were collected in the Xinyuan, Gongliu, Yining and Huocheng Counties of Xinjiang Uighur Autonomous Region in China. Based on phylogenetic analyses and morphological observation, sixty strains of Fusarium accounted for 48% of the total number of fungi isolated from samples were subsequently classified into six species including twenty‐four F. avenaceum, seventeen F. solani, ten F. tricinctum, five F. proliferatum, two F. sporotrichioides and two unfamiliar Fusarium sp. 1. The five previously known species of Fusarium were then tested for pathogenicity to leaves and twigs in vitro. The results indicated that all of the species, except for F. tricinctum, can cause obvious lesions on the leaves of host plants and on the twigs of Fuji and wild apple. This is the first report of Fusarium species pathogenicity in Xinjiang wild apple forests, confirming a new host for these pathogens in this study.     

Phytophthora species associated with dieback of sweet chestnut in Western Turkey

Sweet chestnut (Castanea sativa) is an important tree species in the Marmara and Aegean regions of Turkey as these two regions produce the great majority of edible nuts, especially those used for marron glacé production. Chestnut forests and orchards in these regions showing severe dieback symptoms not associated with chestnut blight were investigated to determine the role of Phytophthora spp. in the decline syndrome. Soil samples were collected from around 108 symptomatic chestnut trees at 29 sites and Phytophthora spp. isolated using soil baiting technique and selective medium. Species isolated were identified by cultural characteristics and ITS sequencing. Phytophthora x cambivora was the dominant species detected in 13 sites, followed by P. cinnamomi (5 sites), P. plurivora (3 sites) and P. cryptogea (1 site). Phytophthora x cambivora was present in both regions, while P. cinnamomi was found only in the Marmara region in coastal areas around Istanbul. When inoculated at the stem bases of 3‐year‐old chestnut saplings, P. cinnamomi produced significantly longer necrotic lesions (7.8–12.0 cm) than P. x cambivora (2.6–6.3 cm) by 12 days after inoculation. Phytophthora plurivora was the least aggressive species causing only small lesions. Phytophthora cryptogea, which represents the first record on chestnut in Turkey, produced intermediate sized lesions in between P. x cambivora and P. plurivora. These results indicate that P. x cambivora and in some areas P. cinnamomi play major roles in the observed dieback of sweet chestnut in western Turkey.     

Incidence of dieback disease following fungal inoculations of sexually and asexually propagated shisham (Dalbergia sissoo)

Shisham (Dalbergia sissoo) is an important multipurpose tree with great economic importance, but this tree has been devastated by dieback disease. Seedlings and asexually propagated (cuttings) plants were artificially inoculated with four fungi (Fusarium solani, Botryodiplodia theobromae, Curvularia lunata and Ganoderma lucidum) to evaluate the potential role of these fungi in shisham dieback disease. Results at 2 years revealed that highest disease was caused by inoculation of F. solani (31.39%), followed by B. theobromae (19.042%) and C. lunata (12.22%), but no dieback disease was caused by G. lucidum. During both years, seedlings exhibited greater susceptibility to disease (17.24%) compared to cuttings (7.83%). In particular, F. solani caused more disease in seedlings (46.18%) compared to cuttings (16.61%). With the F. solani inoculations, maximum disease rate was observed at 8 weeks post‐inoculation both in seedlings (77%) and in cuttings (31%), but the maximum disease increase was observed at 4–5 weeks post‐inoculation. Analysis of variance showed significant differences among the different fungi and also between seedlings and cuttings. F. solani can be considered as a major fungal pathogen contributing to dieback disease of shisham, and asexual propagation can reduce the severity of dieback.     

Characterization of Lasiodiplodia theobromae (Pat.) Griffon & Maubl causing stem canker on Teak (Tectona grandis L.) in Ecuador

Teak is a very important tropical timber in Ecuador. In 2017, teak samples displaying stem canker symptoms were collected in two provinces of Ecuador. From 11 symptomatic trees, 11 isolates resembling a species of Lasiodiplodia were obtained. All isolates obtained induced stem canker on teak plants after artificial inoculation, confirming them as the cause of the observed canker symptoms. Bayesian inference with concatenated sequences of complete ITS and partial TEF-α and β-TUB gene sequences from two representative isolates clustered teak isolates with other sequences of Lasiodiplodia theobromae available in Genbank. This is the first report of L. theobromae causing stem canker on teak plants in Ecuador.     

First report of Sydowia polyspora causing disease on Pinus pinea shoots

The fungus Sydowia polyspora is frequently isolated from conifers worldwide and is considered a pathogen on several hosts. Stone pine (Pinus pinea) is one of the most important forestry species throughout the Mediterranean basin due to the value of the edible pine nut. Stone pines showing tip dieback, needles with tan‐ to yellow‐coloured lesions and shoot death, observed in stands in Portugal, were sampled for analysis. Fungal colonies covered with cream‐coloured spore masses, were consistently obtained. Morphological and phylogenetic analyses of the ITS rDNA region enabled identification of these isolates as S. polyspora. Inoculation tests showed that the fungus caused lesions on excised P. pinea shoots. The symptoms observed might have a negative effect on pine nut production, and thus, evaluation of the impact of this disease is of relevance to future research. This paper is the first to report S. polyspora causing disease on P. pinea.     

First report of Hymenoscyphus fraxineus on Fraxinus excelsior in Montenegro

During the monitoring of the mycological complex on different forest tree species in the Biogradska Gora National Park in north‐east Montenegro, symptoms indicative of ash dieback caused by Hymenoscyphus fraxineus were observed on young Fraxinus excelsior trees in the protected virgin forest, including dieback of plants and branches, wilting of leaves and shoots leading to a “flag‐like” habitus, premature shedding of leaves and longitudinal bark necroses. Using standard isolation methods, slow‐growing cultures with numerous phialides, typical of the asexual phase of the ash dieback fungus, were obtained. In addition, petioles with numerous characteristic apothecia were also recorded. This is the first report of H. fraxineus on common ash in Montenegro. Possible pathways of introduction and implications of the findings are discussed.     

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.     

Decline of Paulownia tomentosa caused by Trametes hirsuta in Serbia

During the monitoring of the health status of nurseries and plantations in Serbia, a decline in a 5‐year‐old Paulownia tomentosa plantation was recorded. Trees displayed symptoms of dieback, massive breaking at different positions, the appearance of decay and fungal fruitbodies at the stems. Using standard isolation methods, white colonies with cottony surfaces and regular growth were obtained from the decaying wood samples, and after the oxidation degree analysis, it was determined that these isolates belonged to Davidson's group 6, indicating white rot basidiomycetes. To identify the isolated fungus, the ITS region of one selected isolate was sequenced. Based on the morphological analysis of the obtained colonies, collected fruitbodies and ITS sequence analysis, this fungus was identified as Trametes hirsuta. This is the first report of T. hirsuta on Paulownia tomentosa in Serbia. Irregularly performed pruning as the possible infection route and the implications of these findings are discussed.     

Land‐use changes influence the sporulation and survival of Phytophthora agathidicida,a lethal pathogen of New Zealand kauri (Agathis australis)

Phytophthora agathidicida is the accepted causal agent of dieback in remnant stands of long‐lived indigenous New Zealand kauri (Agathis australis) and poses a significant threat to the long‐term survival of this species. Little is known about the effect of key soil physicochemical characteristics on the growth of P. agathidicida. In this study, we investigated the growth of P. agathidicida in soils collected from adjacent areas under original kauri forest, short rotation pine (Pinus radiata) plantation forest and grazed pastures. A growth response assay was used to quantify asexual (sporangia) and sexual (oospore) spore counts over 8 days in soils sampled from each land‐use. Significantly higher numbers of sporangia (p < 0.001) and oospores (p < 0.01) were found in pasture and pine forest soil within 2 days of the growth assay trials, suggesting these soils may favour asexual/sexual reproduction in the early stages of P. agathidicida establishment compared to kauri forest soils. Additionally, oospore production significantly increased over 8 days in pine forest soil, suggesting that with an increase in inoculum loads, these soils potentially act as pathogen reservoirs. The soil physicochemical properties (e.g., pH, C and N, phosphorus content and electrical conductivity) investigated in this study did not significantly correspond to spore count data between land‐uses, suggesting that differences in growth response are driven by other edaphic factors not explored in the present study.     

Direct quantitative real‐time PCR assay for detection of the emerging pathogen Neonectria neomacrospora

The epidemic outbreak in northern Europe of Neonectria neomacrospora, the causal agent of dieback in Abies spp., led the European and Mediterranean Plant Protection Organization (EPPO) to include the pathogen on its alert list in 2017. Effective monitoring of this pathogen calls for a rapid and sensitive method of identification and quantification. A probe‐based real‐time PCR (qPCR) assay based on the β‐tubulin gene was developed for the detection and quantification of N. neomacrospora in infected wood samples, and directly for ascospores. This study presents the first published species–specific molecular detection assay for N. neomacrospora. The analytical specificity was validated on taxonomically closely related fungal species as well as on 18 fungal species associated with the host (Abies sp.). The analytical sensitivity was tested on naturally infected wood, on purified pathogen DNA in a matrix of host DNA and on N. neomacrospora ascospores for detection of airborne inoculum. The latter was tested both with a DNA extraction step prior to qPCR and without DNA extraction by direct qPCR on collected ascospores. The assay was specific to N. neomacrospora, with a sensitivity of 130 fg purified DNA, or 10 ascospores by direct qPCR. Omitting DNA extraction and amplifying directly on unpurified ascospores improved assay sensitivity significantly.     

Occurrence and diversity of Phytophthora species in declining broadleaf forests in western Ukraine

In western Ukraine, forest decline and dieback of several broadleaved tree species have become increasingly evident during recent years, and surveys in some areas have shown symptoms indicative of Phytophthora infections. In this study, we aimed to determine the occurrence and diversity of Phytophthora species associated with several broadleaved tree species (Alnus glutinosa, Betula pendula, Castanea sativa, Fagus sylvatica and Quercus robur) from forest stands where dieback has been observed. Rhizosphere soil samples were collected from 14 forest stands during 2017 and 2018 and tested for the presence of Phytophthora species using morphological and molecular methods. Seven Phytophthora species (P. bilorbang, P. cactorum, P. gallica, P. gonapodyides, P. lacustris, P. plurivora and P. polonica), and two other clade six taxa were detected from the various forest types, several of which are probable agents responsible for decline. Four of the Phytophthora species (P. bilorbang, P. gallica, P. plurivora and P. polonica) have previously never been reported from broadleaf forests in Ukraine.     

Pseudomonas associated with Bursaphelenchus xylophilus,its insect vector and the host tree: A role in pine wilt disease?

In this study, we characterized the diversity of Pseudomonas associated with Bursaphelenchus xylophilus, its insect vector (Monochamus galloprovincialis) and its host (Pinus pinaster), by a culture‐independent approach using rpoD clone libraries. Clone libraries of Pseudomonas rpoD were obtained from B. xylophilus, M. galloprovincialis and infected P. pinaster. Most M. galloprovincialis and B. xylophilus sequences grouped together in the P. fluorescens group. Genes related to xenobiotics degradation and phenylacetate synthesis were present in the genomes of the type strains closely related to sequences retrieved from the nematode libraries. Results demonstrated that the nematode, during its life stages inside the tree, maintains a diverse Pseudomonas community that is closely related to the one associated with the insect vector. These bacteria might contribute to degradation of xenobiotics and tree weakening during the nematode tree infection.     

An agglutination test for fast and specific detection of Erwinia psidii

Dieback and wilt, caused by Erwinia psidii (Ep), is one of the most important emergent diseases of Eucalyptus spp. in Brazil. Currently, pathogen detection relies on isolation of bacteria from infected plant tissue and either identification based on morphological, physiological and biochemical tests or DNA amplification using the polymerase chain reaction (PCR), which in many cases is laborious and cumbersome. Considering the need for a simpler and more rapid, yet reliable, method for detecting the pathogen, we obtained a polyclonal antibody (anti‐Ep) and developed an agglutination test for specific detection of E. psidii. The antiserum was produced against the E. psidii strain LPF534 and tested against 101 E. psidii isolates from Eucalyptus spp.; three E. psidii isolates from Psidium guajava; 23 Ralstonia solanacearum and 18 Xanthomonas axonopodis isolates pathogenic to Eucalyptus spp.; and seven endophytic isolates from Eucalyptus spp., three of which are phylogenetically related to the genus Erwinia. Results of direct ELISA indicated that a concentration as low as 3.5 µg/ml of the anti‐Ep antibody was able to detect the E. psidii antigen and that the antibody did not cross‐reacted with other bacteria pathogenic and non‐pathogenic to Eucalyptus spp. In the agglutination test, the anti‐Ep antibody showed positive reaction with all strains of E. psidii tested whereas cross‐reaction with none of the strains that belong to other taxonomic groups was observed. The agglutination test showed a detection limit of 10⁵ colony‐forming units (CFU)/ml, and its specificity was the same as that obtained by PCR amplification using E. psidii‐specific primers. These results demonstrate that the agglutination test developed here is a useful tool for specific, fast and inexpensive detection of E. psidii although only operational on pure bacterial suspensions and not yet directly from infected tissues.     

The first record of Botryodiplodia canker in Poland

This study describes the first observation of Botryodiplodia canker in the Western Carpathians in south‐eastern Poland caused by Botryodiplodia hypodermia (Sacc.) Petr. (syn. Sphaeropsis hypodermia, S. ulmicola). The canker occurred on an approximately 17‐year‐old Ulmus glabra sapling in a mixed conifer/deciduous stand with elm trees severely damaged by Dutch elm disease. This paper describes disease symptoms and provides information on the macro‐ and micromorphology of the fungus isolated from the cankered tissues. The results of BLAST search using DNA sequences obtained for our cultures and subsequent phylogenetic positioning of the fungus among closely related Botryosphaeriaceae indicate that the species is much more closely related to Phaeobotryon than to the other Botryodiplodia or Sphaeropsis species. Moreover, a total of 16 polymorphisms within the ITS region were detected between S. ulmicola associated with Botryodiplodia canker in North America and B. hypodermia associated with the canker observed in Poland. Thus, the “European” variant of “Sphaeropsis” ulmicola can now be easily identified with our barcode sequences. The Botryodiplodia canker is much less prevalent in Europe than in North America. Differences in virulence of “American” and “European” linages and differences in susceptibility of various elm species may be the reason for the higher prevalence of the disease in North America.     

Infection approach of Diplodia sapinea,the causal agent of tip blight of Pinus tabulaeformis in China

To clarify the infection approach of Diplodia sapinea, a pathogen that causes tip blight of Pinus tabulaeformis, the infection process of the pathogen in needles was observed using scanning electron microscopy (SEM). In addition, the disease incidence on branches damaged by Aphrophora flavipes (Hemiptera: Cercopidae) and Dioryctria splendidella (Lepidoptera: Pyralidae) in the forest was also investigated. Then, branches and needles of P. tabulaeformis were inoculated using the D. sapinea spore suspension under indoor and field conditions. The results showed that the damage caused by A. flavipes could aggravate the occurrence of tip blight of P. tabulaeformis to some extent. Moreover, the pathogen could also penetrate 1‐, 2‐ and 3‐year‐old pine needles through stomata in the field. The pathogen infected the 1‐year‐old branches first and then gradually spread to 2‐ and 3‐year‐old branches.     

Bacillus pumilus and Stenotrophomonas maltophilia as two potentially causative agents involved in Persian oak decline in Zagros forests (Iran)

The oak decline is known as one of the most destructive complex diseases causing high economic losses around the world, especially in Iran. The main objective of the present study was to investigate the possible role of bacteria as causative agents of oak decline in the Zagros forests of Iran. To do this, stem, root and leaf samples were taken from symptomatic Persian oak trees (Quercus brantii) in different zones of Zagros forests (Ilam Province, Iran). From 150 bacterial isolates, 20 showed pathogenicity against Geranium seedlings. Among 20 hypersensitivity test positive strains, four strains showed pathogenicity against oak saplings. Based on morphological and 16S rRNA gene sequence analysis, three strains were identified as Bacillus pumilus and one strain as non‐sporulating Gram‐negative Stenotrophomonas maltophilia. Pathogenicity studies of different B. pumilus and S. maltophilia strains revealed that they have potential to cause the disease in oak saplings and symptoms of disorder in Persian oak trees. To our knowledge, there are no previous records of B. pumilus and S. maltophilia causing decline on Fagaceous trees like Q. brantii. More detailed field and molecular studies are required to confirm the absolute role of such bacteria in occurrence of oak decline in Zagros forests.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

Dothistroma spp. in Western Ukraine and Georgia

Dothistroma needle blight (DNB) is among the most serious foliar diseases affecting Pinus spp. globally. Infected needles were collected from potential host species in four locations in western Ukraine and in four locations in eastern Georgia during spring–summer 2015 to update the knowledge on pathogen distribution in these countries. Dothistroma spp. were detected using isolation, sequencing and species‐specific priming (SSPP) PCR. Two new hosts for Dothistroma spp. were recorded in western Ukraine: D. septosporum on Pinus nigra var. australica and D. pini on P. nigra var. mollet. D. septosporum was found on 15‐year‐old P. strobus in western Ukraine. New hosts for D. septosporum were recorded in Georgia on 5‐ to 10‐year‐old naturally regenerated P. sylvestris var. hamata and on 40‐ to 50‐year‐old P. ponderosa trees. D. pini was found for the first time in Georgia on 30‐ to 40‐year‐old P. nigra trees. The work confirmed the presence of both D. septosporum and D. pini in western Ukraine and Georgia, and demonstrated new hosts for both Dothistroma species.