Headspace solid‐phase microextraction gas chromatography–mass spectrometry for the detection of volatile organic compounds released from Ganoderma boninense and oil palm wood

Basal stem rot disease caused by the white‐rot fungus Ganoderma boninense is a major threat to the oil palm industry, and hence, the ability to detect infections at an early stage of development is desired. In this study, a headspace solid‐phase microextraction (HS‐SPME) method coupled with gas chromatography–mass spectrometry (GC‐MS) was employed to analyse the volatile organic compounds (VOCs) released from G. boninense cultures and infected oil palm wood. We examined VOCs released from three types of samples: G. boninense mycelium, oil palm wood and oil palm wood colonized by G. boninense. This preliminary study led to the tentative identification of 57 VOCs, including alcohols, alkanes, volatile acids, ketones, aldehydes, esters, sesquiterpenes and polycyclic aromatic hydrocarbon groups. Aliphatic compounds with eight‐carbon atoms, such as 1‐octen‐3‐ol, 3‐octanone, 1‐octanol and (E)‐2‐octenal, were the most abundant constituents of the Ganoderma samples, whereas furfural and hexanal were the major compounds detected in the oil palm wood samples. Chemometric analyses using cluster heat maps and principal component analyses were used to discriminate between the VOC profiles. The results indicated that the novel method described here could be used to detect Ganoderma disease and, more generally, for chemoecological studies of plant–pathogen interactions.     

Environment-friendly short-term protection of palm wood against mould and rot fungi

Felled palm trunks are susceptible to fungi as long as their moisture content is above fibre saturation. During this period, palm wood has to be protected against mould and rot fungi. The study was aimed at testing environment-friendly organic acids for their protecting efficiency. Small samples of date palm (Phoenix dactylifera) and oil palm (Elaeis guineensis) wood were treated with weak organic acids and subsequently infected by moulds and wood-decay fungi. Short dipping of the samples in solutions of 5% acetic acid and propionic acid, respectively, protected all samples for two months from colonization by Aspergillus niger, Penicillium sp., Cladosporium sp. and by a natural infection. Boric acid (4%) used in practice for protection was ineffective. Decay tests with the white-rot fungus Pleurotus ostreatus, the brown-rot species Coniophora puteana and the soft-rot fungus Chaetomium globosum showed that both acids prevented most samples from fungal colonization for three weeks and reduced the decay considerably during two months.     

Pathogenicity of Malaysian Phytophthora palmivora on cocoa,durian, rubber and oil palm determines the threat of bud rot disease

The epidemic of bud rot disease affecting oil palm in Colombia is primarily caused by Phytophthora palmivora. The pathogen has a cosmopolitan presence that includes Southeast Asia, but to date, bud rot has not been reported in this region. This study provides an overview of the potential risk of Malaysian P. palmivora isolates cross‐infecting other host species, including cocoa, durian, rubber and Malaysian oil palm planting materials (Dura × Pisifera, D × P). On cocoa pods, the durian isolate PP7 caused dark brown necrotic lesions. Detached leaf bioassays showed that P. palmivora isolates PP3 and PP7 infected different hosts, except rubber foliage without wounding. Inoculation tests on cocoa, durian and rubber seedlings caused brown necrotic lesions when stems were wounded, with 10% mortality in cocoa and durian at 17 days post‐inoculation (dpi). However, no further infection was observed, and lesions closed within 14–28 dpi on the non‐wounded seedlings. Pathogenicity tests of oil palm seedlings inoculated with isolates PP3 and PP7 indicated that Malaysian P. palmivora isolates were not pathogenic to oil palms based on localized infection observed only through wounding. Overall, the work demonstrated that Malaysian P. palmivora isolates were able to cross‐infect multiple hosts but did not show severe infections on oil palms.     

Pathogenicity of ophiostomatoid fungi on Picea abies in Slovenia

Pathogenic fungi can survive and develop in living plants, often causing diseases in the host. Some theories speculate that pathogenic ophiostomatoid fungi provide benefits to its vectors – bark beetles – by overcoming the tree's defence mechanisms. This study reports the results of an experiment in south‐eastern Europe in which mature and seedling Norway spruce trees were artificially inoculated with various ophiostomatoid fungi. The aim of the experiment was to determine the relative virulence of ophiostomatoid fungi by assessing the ability of the fungi to stimulate host tree defence mechanisms through inoculation experiments. Experiments were performed by inoculation of Picea abies in seedling and mature trees. The following fungi were used in low‐density and seedling inoculations: Ophiostoma ainoae, O. brunneo‐ciliatum, Grosmannia cucullata and an unidentified Leptographium sp., O. bicolor, O. fuscum, O. piceae, G. penicillata and G. piceiperda. Endoconidiophora polonica was used in mass and seedling inoculations. Various characteristics such as host vitality, blue stain, lesion and resin outflow were measured before and after the trees were felled. E. polonica caused blue stain, induced large lesions and killed some of the mature trees and seedlings, confirming earlier reports that it is a strong wound pathogen. Only E. polonica, Leptographium sp. and O. ainoae caused blue stains in the sapwood of inoculated seedlings. In low‐density inoculations, G. piceiperda induced intense necrosis and had higher values for all the characteristics monitored. Some of the other ophiostomatoid fungi showed a moderate level of pathogenicity. Fungi with the capacity to stimulate a host defence mechanism could play a role in the establishment of bark beetle populations.     

Relationships between Oak powdery mildew incidence and severity and commensal fungi

Oak (Quercus robur) powdery mildew is a common and damaging fungal disease. In a local survey at Reading, UK, oak powdery mildew was common on trees of all height classes but was most common on trees of 3–9 m. A variety of other fungal species were commonly found growing in association with oak powdery mildew colonies. The abundance of such fungi was estimated through stratified sample surveys for 2.5 years. The taxa most commonly associated with oak powdery mildew were Acremonium sp., Trichoderma sp., Ampelomyces/Phoma sp. and Leptosphaerulina australis. Nearly 90% of mildew colonies were associated with L. australis, which is not generally considered as a mycoparasite or antagonist, in contrast with the other three fungi. Abundance varied between June and October surveys. Acremonium sp. abundance was greater in summer samplings, whereas L. australis and Trichoderma sp. abundances were greater in autumn samplings. Ampelomyces/Phoma sp. was never observed in the absence of powdery mildew. Relationships between the mildew‐associated fungi and oak powdery mildew appeared curved and differed significantly between sampling years. L. australis was positively correlated with the other three associated fungi studied when powdery mildew was also present . The variety and high population densities of the mildew‐associated fungi suggest that they may be important in determining the final density of oak mildew and the damage caused by it.     

Mycobiota in needles of Abies alba with and without symptoms of Herpotrichia needle browning

The ascomycete Nematostoma parasiticum (syn.: Herpotrichia parasitica) is commonly perceived as the causal agent of the so‐called Herpotrichia needle browning in silver fir (Abies alba). However, its fruitbodies are rarely present on symptomatic needles, which are also colonized by many presumably saprotrophic fungi. We compared the internal colonization of healthy and symptomatic needles on two sites in Poland. In addition, the endophytic mycobiota in needles of various age was recorded on two other sites without disease symptoms. Fungi were isolated from 95.6% of the dead needles and from 62.9% of the living needles on symptomatic trees, whereas on healthy trees, only 45.0% of the needles were colonized internally. Colonization frequency increased with needle age. From a total of 2017 isolates, 116 fungal taxa were identified. Frequency of many species was influenced by needle type. Anthostomella formosa, Gloeosporidiella sp., Hypoxylon fragiforme, Xylaria hypoxylon and X. polymorpha were the most common fungi isolated from living needles. In symptomless living needles, fungi occurred significantly more often in the basal than in the apical parts. In dead needles, the most common fungi were Alternaria alternata, Paraconiothyrium sporulosum, Fusarium sp., Mollisia cinerea, Rhizoctonia sp., Rhizosphaera oudemansii, Thysanophora penicillioides, Xylaria hypoxylon and X. polymorpha. Rhizoctonia sp. was the most frequently isolated fungus in dead needles (23.4%) but occurred rarely also in living needles (0.3–1.1%). The supposed pathogen N. parasiticum was detected only sporadically (at most in 0.6% of the needles). Our findings demonstrate the need for understanding the role of Rhizoctonia sp. in Herpotrichia needle browning disease aetiology.     

Cross‐infectivity of oil palm by Phytophthora spp. isolated from perennial crops in Malaysia

Bud rot disease or “Pudricion del cogollo” (PC) of oil palm is a major constraint on production in Colombia and neighbouring countries such as Brazil, Costa Rica, Ecuador, Nicaragua, Panama, Peru and Surinam. To date, there are no documented reports of Phytophthora disease of oil palm in South‐East Asia. This research, therefore, was conducted to determine the pathogenic potential of Phytophthora palmivora and Phytophthora nicotianae on oil palm using both in vitro and nursery inoculation experiments. In vitro inoculation of both P. palmivora and P. nicotianae on immature oil palm leaflets caused discoloration within 2 days of inoculation and incubation at 25 ± 1.5°C, 100% RH. Similarly, in nursery trials, lesions formed on the buds (unopened leaflets) 3 days after inoculation with P. palmivora or P. nicotianae zoospore suspensions. No lesions developed on untreated leaflets in either in vitro or nursery inoculation experiments. Phytophthora spp. were re‐isolated from leaflet lesions and confirmed as the inoculated pathogens.     

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.     

Newly recognized bacteria involved in trunk scab and canker of white and hybrid poplars

In a 4‐year‐old collection of native Italian Populus alba, growing in the eastern Po valley, many trunks showed severe symptoms characterized by brown spots, similar to trunk scab and/or by cankers. Canker‐like growths, often with gall‐like‐formations in the earlier developmental phases were also found on some clones of P. trichocarpa and their hybrids, as well as on P. deltoides growing in several Northern Italian nurseries. Bacteria and fungi present in the affected tissues and potentially involved in the disease were examined. No fungi were detected, however, several species of bacteria were isolated and identified: Pseudomonas mendocina and Erwinia herbicola group, from the brown spots; Erwinia carotovora sub sp. carotovora and, occasionally, E. herbicola group, from cankers. Only E. carotovora sub sp. carotovora produced clear symptoms of canker when artificially inoculated on young plants of different poplar species. This is the first time this symptomology on poplar species has been clearly related to E. carotovora sub sp. carotovora.     

Fungi associated with Cyclaneusma needle cast in Scots pine in the west of Poland

Needles of Pinus sylvestris with and without symptoms of Cyclaneusma needle cast, from the west of Poland, were examined for abundance and diversity of fungi using Illumina sequencing. Fungal communities were dominated by Ascomycota (93.6%–98.6% of OTUs). Basidiomycota and non‐culturable fungi were less frequent. Needles were colonized by 260 taxa. Ascomycota, Basidiomycota and Glomeromycota were represented by 149, 39 and 1 taxa. Abundance of fungi was least in 1.5‐year‐old needles attached to twigs and greatest in fallen 2‐year‐old needles. Fungal communities had least diversity in 1.5‐year‐old needles and most diversity in 0.5‐year‐old needles of current growth. It was found that (a) the most common fungi were the needle pathogens Cyclaneusma minus, Lophodermium spp. and Sydowia polyspora; (b) less common potential pathogens were Cenangium ferruginosum, Coniothyrium complex, Desmazierella acicola, Neocatenulostroma germanicum and species in the genera Neodidymelliopsis, Pestalotiopsis, Phoma, Pleurophoma and Pyrenochaeta; (c) common primary or secondary saprotrophs included species of Alternaria, Aureobasidium, Beauveria, Cladophialophora, Cladosporium, Epicoccum, Exophiala, Lecanicillium, Penicillium, Cryptococcus and Kwoniella; (d) Lophodermium was represented mostly by Lophodermium pinastri which occurred 4–72 times more frequently than Lophodermium seditiosum; (e) frequencies of C. minus and C. ferruginosum were lower in the 0.5‐year‐old symptomless needles, increased in the symptomatic and symptomless 1.5‐year‐old needles and decreased after needle fall; (f) frequency of L. seditiosum was highest in 0.5‐year‐old needles; (g) frequency of L. pinastri increased with needle age whereas S. polyspora increased after needle fall; (h) lower frequency of L. pinastrii was associated with higher frequency of S. polyspora. It was concluded that Cyclaneusa needle cast in Poland may be caused by C. minus accompanied by C. ferruginosum, L. seditiosum, L. pinastrii and S. polyspora. Participation of Coniothyrium spp., Epicoccum nigrum, Pestalotiopsis spp. and Phoma spp. in the disease progress cannot be excluded.     

Impacts of inoculation with Herpotrichia pinetorum,Gremmenia infestans and Gremmeniella abietina on Pinus nigra subsp. pallasiana and Cedrus libani seedlings in the field

Herpotrichia pinetorum, Gremmenia infestans and Gremmeniella abietina were inoculated onto 2‐year‐old Anatolian black pine (Pinus nigra subsp. pallasiana) and Taurus cedar (Cedrus libani) seedlings planted in a high mountain forest (1800 m a.s.l) in south‐western Turkey, to determine the effects of these fungi during winter. In June, 8 months after inoculation, 39.9% of experimental plants were dead and 20.4% of the surviving plants failed to flush. Gremmeniella abietina and H. pinetorum caused the most fatalities. Prevention of new shoot formation on surviving plants, however, was mainly an effect of G. abietina infections, although many surviving plants inoculated with G. infestans or H. pinetorum also failed to flush. All three pathogens had the potential to severely damage young plants of P. nigra subsp. pallasiana and C. libani growing at high elevations near to forests with heavy inoculum loads. The implications of this finding for P. nigra afforestations at high altitudes in Turkey are discussed. This study is also the first to report that G. infestans can infect and cause disease on young C. libani plants.     

Root lesions in large loblolly pine (Pinus taeda L.) following inoculation with four root‐inhabiting ophiostomatoid fungi

Loblolly pine decline, characterized by deteriorating root systems leading to shortening and thinning of foliage, has been observed throughout portions of the south‐eastern United States. Several root‐inhabiting ophiostomatoid fungi, including Leptographium procerum, Leptographium terebrantis, Leptographium serpens, and Grosmannia huntii are associated with lateral root damage on declining loblolly pine. Trees of various ages were inoculated in primary lateral roots during fall (2006 and 2007) and spring (2007 and 2008). All fungi caused a darkened, resin‐filled lesion on the surface of the phloem, extending into the xylem that was larger than that of controls. Only lesions associated with G. huntii infection were significantly larger in the spring season, compared with the fall. Grosmannia huntii was found to be the most virulent fungus, causing lesions that were longer, deeper and larger than all other fungal species during the spring and larger than L. terebrantis and L. procerum in the fall. Leptographium serpens was the second most virulent fungal species, causing lesions larger than L. procerum and L. terebrantis (with the exception of lesion depth) during both seasons. These tests indicate that G. huntii and L. serpens are significant root pathogens, capable of causing considerable damage, while L. terebrantis and L. procerum may be less virulent. Depending on the actions of their vectors, G. huntii and L. serpens may be responsible for significant root deterioration and tree disease.     

Effect of host plants on development and reproduction of <Emphasis Type="Italic">Rhynchophorus ferrugineus</Emphasis> (Olivier) (Coleoptera: Curculionidae)

The development, survivorship and reproduction of red palm weevil Rhynchophorus ferrugineus, reared on five ornamental palm slices, were studied in the laboratory at constant temperature of 26°C. The developmental time of R. ferrugineus was 68.8 days on Washington palm (Washingtonia filifera), 74.1 days on Canary Island date palm (Phoenix canariensis), 82.1 days on Chusan palm (Trachycarpus fortunei), 85.4 days on pindo palm (Butia capitata) and 90.6 days on silver date palm (Phoenix sylvestris), respectively, and the developmental time on silver date palm was significantly longer than that on the other plants. The survival of immature R. ferrugineus ranged from 25.0 to 38.3%. Most mature larvae began to pupate at eighth-instar on Canary Island date palm and Washington palm, while on Chusan palm, Pindo palm and silver date palm, most of them began to pupate at ninth-instar. The mean lifetime fecundity of R. ferrugineus on Canary Island date palm, Chusan palm, Pindo palm, Washington palm and silver date palm was 267.8, 134.0, 109.8, 216.0, and 131.4 eggs, respectively, and lifetime fecundity on Canary Island date palm and Washington palm was significantly greater than that on the other plants. The intrinsic rate of natural increase (r _m ), net reproductive rate (R ₀) and mean generation time (T) were 0.038, 78.3 and 115.0 days on Canary Island date palm, 0.028, 33.1 and 125.5 days on Chusan palm, 0.029, 40.9 and 128.2 days on Pindo palm, 0.041, 64.0, 101.4 days on Washington palm, and 0.025, 30.6, 135.6 days on silver date palm, respectively. Based on population growth parameters, it is concluded that Canary Island date palm and Washington palm were the more suitable host plants and silver date palm was the least suitable host plant for R. ferrugineus.     

Intraspecific response of Pinus taeda L. to Grosmannia huntii and Leptographium terebrantis infection

We examined intraspecific and inter‐year variation in tolerance of Pinus taeda to two ophiostomatoid fungi, Leptographium terebrantis and Grosmannia huntii. Containerized seedlings of P. taeda from 27, 32, 17 and 23 different elite genetic families were artificially inoculated with L. terebrantis and G. huntii in years 2013, 2014, 2016 and 2017, respectively. Six connector families were inoculated every year. Eight weeks post‐inoculation, lesion and occlusion were measured on each seedling to determine the relative susceptibility/tolerance of families to these fungi. Pinus taeda families widely differed in these parameters suggesting intraspecific variation in the susceptibility/tolerance to the inoculated pathogens. The overall tolerance of the connector families to these fungi varied among the experimental years. These results showed that intraspecific variation to L. terebrantis and G. huntii exists among P. taeda families and it could be possible to select tolerant families to minimize the potential impact due to these fungi.     

Identification of wood‐decay fungi and assessment of damage in log depots of Western Black Sea Region (Turkey)

The aim of this study was to determine and quantify the wood‐decay fungi found on logs of forest tree species (beech, oak, hornbeam, Scots pine and fir) stored in log depots located in six different provinces in the Western Black Sea Region of Turkey. Additionally, it was aimed to determine the natural durability of some important wood species against the most commonly detected wood‐decay fungi. Eighteen families, 31 genera and 45 species belonging to the division Basidiomycota were detected; Antrodia crassa was identified for the first time in Turkey. The abundance of Panus neostrigosus, Polyporus meridionalis, Trametes hirsuta, T. versicolor and Stereum hirsutumincreased significantly with the holding time of the logs (r = 0.99, 0.87, 0.53, 0.57 and 0.78, respectively, p < 0.05). The majority of the fungal species were detected on logs stored in depots for 4–6 years (66%). The percentage of fungal species found on the logs with a holding time of three years or less was 29%, whereas the percentage for those detected on logs stored for seven or more years was 31%. Among the wood species, the greatest number of fungal species (29) and highest amount of fungi (2,539) occurred on beech wood. Natural durability tests showed that T. versicolor caused the greatest loss of wood mass, with an average of 23%. Field studies and natural durability tests performed in the laboratory showed that beech wood lost the most mass among the timber species studied.     

Root‐colonizing ophiostomatoid fungi associated with dying and dead young Scots pine in Poland

Ophiostomatoid fungi are carried by various bark beetles. However, very little is known about the role of these fungi in conifer roots. We studied ophiostomatoid fungi in roots of dying and dead Pinus sylvestris trees and tested the potential phytotoxicity of some isolates using a sensitive bioassay with Lepidium sativum in Poland. Fungi were identified based on their morphology and DNA sequencing. Three ophiostomatoid fungi, Leptographium procerum, Sporothrix inflata and Ophiostoma pallidulum, were isolated from the roots. The most abundant soil‐borne fungus, S. inflata, and relatively rare O. pallidulum were isolated for the first time from roots of dying and dead pine trees. The frequency of S. inflata and O. pallidulum correlated with tree decline. The fungi were isolated more frequently from roots of dead than dying trees. Sporothrix inflata and O. pallidulum slightly reduced the stem and root growth of L. sativum. Leptographium procerum reduced more significantly root than stem growth. This species reduced root elongation 32–54% after 10–17 days of incubation.     

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.     

Phylogenetic and pathogenicity evaluation of the marasmioid fungus Marasmius palmivorus causing fruit bunch rot disease of oil palm

Marasmius palmivorus is a marasmioid fungal species that exhibits parasitic behaviour, although most marasmioids are rarely parasitic. The fungus has been reported to cause fruit bunch rot disease of oil palm and coconut, but only a few studies on its pathogenic behaviour are available, particularly on oil palm. Hence, there is a need to assess the ability of the fungus to act as a pathogen and to study its molecular evolution and taxonomy. Nine isolates of M. palmivorus were successfully isolated from basidiocarps and diseased fruitlets of oil palm collected from oil palm plantations and were morphologically characterized on potato dextrose agar (PDA) followed by molecular identification based on nucleotide sequence alignments of internal transcribed spacer (ITS) regions of ribosomal RNA (rRNA) gene clusters with sequences from GenBank. Koch's Postulates confirmed that M. palmivorus could infect oil palm fruitlets with symptoms similar to those observed for bunch rot disease. Molecular phylogenetic studies using nucleotide sequences of ITS and the nuclear ribosomal large subunit (nLSU) showed that isolates sharing the same phenotypic characteristics of Marasmius palmivorus and Marasmiellus palmivorus are monophyletic and share a common ancestor. The fungus has also been shown to be more closely related to the genus Marasmius than Marasmiellus; therefore, we support retention of the taxon name of the pathogen causing bunch rot disease of oil palm as Marasmius palmivorus.     

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.     

Interactions between callus cultures of Pinus sylvestris and pine fungi with different trophic properties

Fungal virulence may be studied using tissues cultures of host plants in dual cultures in vitro, enabling analyses of interactions with undifferentiated cells of their host plants. Three genotypes of Pinus sylvestris callus, initiated by somatic embryogenesis, were used for establishing dual cultures with fungi pathogenic, endophytic or saprotrophic on pine needles or shoots. Fungal growth towards the plant callus tissue differed, depending on the life strategy of the fungus. The pathogen Gremmeniella abietina proved the slowest colonizer of callus whereas the saprotrophic Phacidium lacerum was the fastest. Gremmeniella abietina partially overgrew the callus, causing extensive necrosis and death within 10 days after inoculation. Anthostomella formosa, an endophyte of pines, did not cause evident symptoms of callus degradation: after 10 days of dual culture, the callus cells remained greenish and at least 50% of cells were alive. In dual cultures Ph. lacerum, callus remained alive until the end of the experiment, maintaining a white‐creamy colour with a loose cell structure. Electrophoresis of protein extracts from the callus showed the presence of additional bands of 25–35 kDa only in host tissues challenged with the pathogen G. abietina, possibly indicating the production of pathogenesis‐related proteins. This work has shown that pine callus does not respond equally to challenge with different fungal isolates. In general, one‐third of the isolates of each fungus examined showed greater virulence compared to other isolates.