Differential effects of plant ontogeny and damage type on phloem and foliage monoterpenes in jack pine (Pinus banksiana)

Coniferous trees have both constitutive and inducible defences that deter or kill herbivores and pathogens. We investigated constitutive and induced monoterpene responses of jack pine (Pinus banksiana Lamb.) to a number of damage types: a fungal associate of the mountain pine beetle (Dendroctonus ponderosae Hopkins), Grosmannia clavigera (Robinson-Jeffrey & R.W. Davidson); two phytohormones, methyl jasmonate (MJ) and methyl salicylate (MS); simulated herbivory; and mechanical wounding. We only included the fungal, MJ and mechanical wounding treatments in the field experiments while all treatments were part of the greenhouse studies. We focused on both constitutive and induced responses between juvenile and mature jack pine trees and differences in defences between phloem and needles. We found that phytohormone applications and fungal inoculation resulted in the greatest increase in monoterpenes in both juvenile and mature trees. Additionally, damage types differentially affected the proportions of individual monoterpenes: MJ-treated mature trees had higher myrcene and β-pinene than fungal-inoculated mature trees, while needles of juveniles inoculated with the fungus contained higher limonene than MJ- or MS-treated juveniles. Although the constitutive monoterpenes were higher in the phloem of juveniles than mature jack pine trees, the phloem of mature trees had a much higher magnitude of induction. Further, induced monoterpene concentrations in juveniles were higher in phloem than in needles. There was no difference in monoterpene concentration between phytohormone applications and G. clavigera inoculation in mature trees, while in juvenile trees MJ was different from both G. clavigera and simulated herbivory in needle monoterpenes, but there was no difference between phytohormone applications and simulated herbivory in the phloem.     

Mountain pine beetle-associated blue-stain fungi are differentially adapted to boreal temperatures

Mountain pine beetles (MPB) are the most serious pest of lodgepole pine in Canada and are likely to invade boreal jack pine forests. MPB vector three blue-stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to beetle success. Fungal survival at extreme boreal temperatures will contribute to their success in jack pine. Growth, sporulation and survival of the three fungi at −20 to 37°C were tested in vitro. Overwintering survival of G. clavigera and O. montium was assessed in vivo. All species grew at 5–30°C, with optimal growth at 20–25°C. Grosmannia clavigera and L. longiclavatum survived at −20°C, but O. montium died. Growth of G. clavigera and L. longiclavatum was inhibited at 30°C, but O. montium grew well. Grosmannia clavigera and O. montium overwintered in living pines. These results suggest that G. clavigera and L. longiclavatum were adapted to cold boreal winters but not hot summers, with the converse true for O. montium. Temperature tolerance varied among G. clavigera isolates. British Columbian and Californian isolates grew faster at 25°C than Albertan isolates. Isolates from Alberta and Idaho/Montana grew optimally at 20°C, while British Columbian and Californian isolates grew optimally at 25°C.     

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.     

Biochemical responses associated with induced resistance to Colletotrichum acutatum in Pinus radiata seedlings treated with methyl jasmonate and Trichoderma spp.

The effect of Trichoderma (T. atrobrunneumFCC320 and T. atrovirideLU633) and/or methyl jasmonate (MJ) on resistance to terminal crook (Colletotrichum acutatum) and on seedling biochemistry was investigated in radiata pine (Pinus radiata) seedlings. Seedlings were germinated and grown in Trichoderma‐amended or non‐amended media for 3 months and then sprayed with 2.25 mM MJ 1 week before inoculation with C. acutatum. The incidence and severity of terminal crook in the seedlings treated with MJ and Trichoderma+MJ were lower than in Trichoderma‐treated and Trichoderma‐untreated seedlings. The MJ‐induced resistance response was concomitant with an increase in the concentrations of the monoterpenes α‐pinene, β‐pinene, β‐phellandrene, camphene and myrcene in needles, and also α‐pinene, β‐pinene and camphene in stems. The concentrations of α‐pinene, β‐pinene and camphene were elevated from at least 1 week until 4 weeks after MJ application, compared with those in non‐MJ counterparts. Trichoderma alone did not affect monoterpenes, but the concentrations of α‐pinene, β‐pinene and camphene were greater in needles of Trichoderma+MJ than in MJ‐treated seedlings after 28 days. Total phenolic concentration in needles and peroxidase activity in stems were twofold greater in MJ‐treated seedlings than in non‐MJ seedlings over the same period. None of the treatments affected the activity of peroxidase in needles. It is proposed that the accumulation of monoterpene and phenolics and the induction of peroxidases contribute, in part, to MJ‐induced resistance to terminal crook in radiata pine seedlings.     

A comparison of heat pulse velocity and lesion lengths for assessing the relative virulence of mountain pine beetle‐associated fungi on jack pine

The mountain pine beetle (MPB) vectors three blue‐stain fungi, Grosmannia clavigera, Ophiostoma montium and Leptographium longiclavatum, which contribute to the success of the beetles and the death of the trees. The utility of two methods, heat pulse velocity (HPV) and lesion length, for assessing the relative virulence of these fungi were compared on jack pine in central Alberta. The HPV monitoring apparatus failed to detect xylem sap flow in any of the trees and, thus, could not be used to assess fungal virulence. In contrast, measurement of lesion lengths was more sensitive and provided further evidence that G. clavigera and L. longiclavatum are more virulent than O. montium. The failure of the HPV apparatus to detect sap flow suggests that the study trees were moisture stressed, a factor likely to increase their susceptibility to MPB. Thus, this method is not appropriate for assessing the response of the most susceptible (i.e. drought stressed) trees to MPB and its associated fungi.     

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.     

Mountain pine beetle‐associated blue‐stain fungi in lodgepole × jack pine hybrids near Grande Prairie,Alberta (Canada)

The mountain pine beetle (MPB), the most serious pest of lodgepole pine in mountainous western Canada, spread northeastward into lodgepole × jack pine hybrids in the boreal forest of Alberta in 2006. The MPB vectors three species of blue‐stain fungi, which contribute to the success of the beetles. These fungi were isolated from MPB larvae and galleries in several lodgepole × jack pine stands in the Grande Prairie region of northwestern Alberta in autumn 2006 and winter and spring 2007. Fungi were recovered from more than 95% of gallery systems. The three fungi were similarly prevalent but Ophiostoma montium was the most frequently isolated fungus at each sampling point, isolated from 72% to 90% of gallery systems compared with 63% to 78% for Grosmannia clavigera, and 61% to 86% for Leptographium longiclavatum. Ophiostoma montium and G. clavigera were isolated from more larvae than gallery samples, with the opposite true for L. longiclavatum. Most gallery systems contained multiple fungi with three fungi per gallery system being more common in autumn and winter and two fungi more common in the spring. The combination of G. clavigera and L. longiclavatum was less common among gallery systems with two fungi than either of the pairwise combinations containing O. montium. Fungal prevalence was the same above and below snow level. The prevalence of the three fungi did not differ significantly among stands sampled in the spring but stands with more G. clavigera tended to have less L. longiclavatum. The winter of 2006–2007 was colder than average throughout Alberta with temperatures below ?30°C in November, January and February, and all three fungi were present after the cold winter while most larvae had died, suggesting that overwintering mortality of the fungi will not limit persistence and spread of MPB in the boreal forest.     

A new Leptographium species from the roots of declining Pinus sylvestris in Switzerland

Scots pine (Pinus sylvestris) trees have been declining in the Rhône valley, Switzerland, for almost three decades. In an assessment of the role of fungi in this syndrome, the dominant fungus isolated from stained roots was a Leptographium species, morphologically similar to the asexual state of Grosmannia serpens. We examined isolates of this fungus based on DNA sequences of four protein‐coding genes including actin, β‐tubulin, calmodulin and translation elongation factor‐1 alpha. The results showed that they were of a distinct, undescribed taxon related to species in the Grosmannia serpens and G. wageneri complexes. The fungus, described here as Leptographium rhodanense sp. nov., resembles other species in the two species complexes morphologically, and most probably ecologically, as is suggested by the fact that it was isolated from stained pine roots.     

Antifungal properties of terpenoids in Picea abies against Heterobasidion parviporum

The antifungal properties of 14 major oleoresin‐constitutive terpenoids of Norway spruce (Picea abies) against Heterobasidion parviporum were evaluated in vitro at three gradient concentrations, 0.1, 0.2 and 0.4 μmol/cm², on potato dextrose agar medium. Eight monoterpene hydrocarbons (+)‐ and (?)‐α‐pinene, (?)‐β‐pinene, (+)‐3‐carene, myrcene, (+)‐ and (?)‐limonene, terpinolene; four oxygenated monoterpenes α‐terpineol, terpinen‐4‐ol, 1,8‐cineole, bornyl acetate; and two resin acids abietic acid and dehydroabietic acid were selected. Abietic and dehydroabietic acids showed the highest antifungal activities at all concentrations. Among oxygenated monoterpenes, bornyl acetate and α‐terpineol showed antifungal activity at the highest concentration. Among monoterpene hydrocarbons, (+)‐α‐pinene showed similar activity to terpinen‐4‐ol and 1,8‐cineole at the highest concentration; however, it was lower than α‐terpineol and bornyl acetate. Other monoterpene hydrocarbons inhibited mycelial growth by <10%. Re‐extraction of medium surfaces after the test period revealed that most α‐terpineol and terpinen‐4‐ol remained unchanged on the surface but monoterpene hydrocarbons completely disappeared. However, notable fungal transformed products were observed on surfaces applied with 1,8‐cineole and bornyl acetate. Thus, mycelial growth inhibition of monoterpenoids might be caused by complex functions such as biodegradation and/or detoxification by interaction between mycelium and compound. These results provide a basis for future studies considering the role of chemodiversity in the comprehensive chemical defence of P. abies.     

Genetic diversity of the myrtle rust pathogen (Austropuccinia psidii) in the Americas and Hawaii: Global implications for invasive threat assessments

Since the myrtle rust pathogen (Austropuccinia psidii) was first reported (as Puccinia psidii) in Brazil on guava (Psidium guajava) in 1884, it has been found infecting diverse myrtaceous species. Because A. psidii has recently spread rapidly worldwide with an extensive host range, genetic and genotypic diversities were evaluated within and among A. psidii populations in its putative native range and other areas of myrtle rust emergence in the Americas and Hawaii. Microsatellite markers revealed several unique multilocus genotypes (MLGs), which grouped isolates into nine distinct genetic clusters [C1–C9 comprising C1: from diverse hosts from Costa Rica, Jamaica, Mexico, Puerto Rico, and USA‐Hawaii, and USA‐California; C2: from eucalypts (Eucalyptus spp.) in Brazil/Uruguay and rose apple (Syzygium jambos) in Brazil; C3: from eucalypts in Brazil; C4: from diverse hosts in USA‐Florida; C5: from Java plum (Syzygium cumini) in Brazil; C6: from guava and Brazilian guava (Psidium guineense) in Brazil; C7: from pitanga (Eugenia uniflora) in Brazil; C8: from allspice (Pimenta dioica) in Jamaica and sweet flower (Myrrhinium atropurpureum) in Uruguay; C9: from jabuticaba (Myrciaria cauliflora) in Brazil]. The C1 cluster, which included a single MLG infecting diverse host in many geographic regions, and the closely related C4 cluster are considered as a “Pandemic biotype,” associated with myrtle rust emergence in Central America, the Caribbean, USA‐Florida, USA‐Hawaii, Australia, China‐Hainan, New Caledonia, Indonesia and Colombia. Based on 19 bioclimatic variables and documented occurrences of A. psidii contrasted with reduced sets of specific genetic clusters (subnetworks, considered as biotypes), maximum entropy bioclimatic modelling was used to predict geographic locations with suitable climate for A. psidii which are at risk from invasion. The genetic diversity of A. psidii throughout the Americas and Hawaii demonstrates the importance of recognizing biotypes when assessing the invasive threats posed by A. psidii around the globe.     

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.     

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.     

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.     

An integrative study of Sakia sisanganensis n. sp. (Rhabditida: Tylenchidae) from Sisangan forest,Iran, and new morphological observations for the genus

Further data on the morphology (the lip region characters) and phylogeny of the genus Sakia are presented. The new observations were based on scanning electron microscopy (SEM) and light microscopy (LM). A new species, Sakia sisanganensis n. sp., was recovered from rotten wood of a dead beech tree (Fagus orientalis) in northern Iran, herein described and illustrated based on an integrative approach, that is morphological, morphometric and molecular characters. The new species is characterized by a combination of the following features: fine transverse striae and vestigial single band in the lateral field in SEM. Labial area dorso‐ventrally flattened. Oral region with two concentric hexagonal plates, the inner one apparently elevated. Amphidial openings short, slit‐like. Stylet delicate. Median bulb fusiform to spindle‐shaped with weak valvular apparatus. Spermatheca functional. Tail filiform with faintly pointed tip and males common. The new species was morphologically compared with four known species of the genus, viz., S. alii, S. arboris, S. castori and S. indica, all having indistinct lateral fields. Molecular phylogenetic analyses were performed based on the small subunit ribosomal RNA gene (SSU rDNA). In the Bayesian inference (BI), S. sisanganensis n. sp. with two isolates was strongly supported as a sister taxon of a clade harbouring S. arboris + Lelenchus species. However, in the maximum likelihood (ML) analysis, the new species formed a clade with S. arboris, thus supporting the reciprocal monophyly of the genera Sakia and Lelenchus. Accordingly, the test of monophyly was performed (using Bayes factor) and the results did not reject the monophyly of sakia (i.e., S. sisanganensis n. sp. and S. arboris as sister taxa) based on the currently available data.     

Intraspecies variation of mature Pinus taeda in response to root‐infecting ophiostomatoid fungi

Seedling screening studies have shown intraspecies variation in susceptibility of Pinus taeda (loblolly pine) to Leptographium terebrantis and Grosmannia huntii, the causal agents of root infection in Pinus species. However, it is critical to understand the susceptibility of mature P. taeda trees. Roots of mature P. taeda families determined as susceptible and tolerant to L. terebrantis and G. huntii by previous seedling screening trials were artificially inoculated with the same fungal isolates. Dark necrotic lesion and vascular occlusion were recorded 8 weeks later. Families previously considered as susceptible had relatively longer lesions and occlusions. The variation in susceptibility to the two fungi remained the same as exhibited by families in the seedling trial. These results suggest intraspecies variation in relative susceptibility of P. taeda to L. terebrantis and G. huntii remains similar regardless of the age of the tree.     

Hyptis suaveolens and Hyptis spicigera (Lamiaceae) essential oils: qualitative analysis, contact toxicity and repellent activity against Sitophilus granarius (L.) (Coleoptera: Dryophthoridae)

Essential oils from Hyptis suaveolens (L.) Poiteau (Lamiaceae) and Hyptis spicigera Lamarck (Lamiaceae) were first analysed by gas chromatography and by gas chromatography/electron impact mass spectroscopy and then evaluated for toxicity and repellent activity against Sitophilus granarius (L.) (Coleoptera: Dryophthoridae), one of the most serious worldwide stored grain pests. Fifty-six compounds have been identified in the essential oil of H. suaveolens: monoterpene hydrocarbons were the most represented volatiles (64.1%), followed by sesquiterpene hydrocarbons (24.0%), oxygenated monoterpenes (8.1%) and oxygenated sesquiterpenes (2.4%). Diterpenes and non-terpene derivatives were scarcely represented. Sixty compounds have been identified in the essential oil of H. spicigera: monoterpene hydrocarbons were the most represented class of volatiles (70.4%), followed by sesquiterpene hydrocarbons (22.6%). Results from topical applications on insects showed that both essential oils had an effective insecticidal activity. The complete kill of S. granarius was observed 24 h after treatment at a minimum effective dose of 0.4 and 0.6 μl per insect with H. suaveolens and H. spicigera oil, respectively. Results from repellency tests showed that the two essential oils had a repellent activity on S. granarius adults: at the lowest dose (2 × 10⁻⁴ μl oil per cm²), H. spicigera essential oil exhibited a higher repellent effect in comparison to H. suaveolens. No significant differences were observed for the repellent effect between the two essential oils at the highest dose (2 × 10⁻² μl oil per cm²).     

Ophiostomatoid fungi isolated from three different pine species in Argentinian Patagonia

Pine plantations in Argentinian Patagonia cover ca. 95,000 ha in Chubut, Río Negro and Neuquén provinces. Exotic bark beetles (Orthotomicus laricis, Hylastes ater and Hylurgus ligniperda) commonly occur in freshly cut logs, stumps and slash. These beetles are vectors of “ophiostomatoid” fungi which include primary tree pathogens as well as important agents of blue stain. The aim of this study was to identify these beetle‐associated fungi. Sawing mills and pine plantations were surveyed three consecutive years. Fungal isolates from stained logs, processed wood and insect galleries were identified based on morphological and DNA sequence comparisons of ITS and β‐tubulin gene regions. Two Grosmannia, one Graphilbum and three Ophiostoma species were identified. Ophiostoma piliferum and O. peregrinum sp. nov. were the most frequently isolated taxa. O. peregrinum occurred in all provinces, colonizing different conifer species and, interestingly, also the native broadleaved species Nothofagus dombeyi. Pine plantation forestry in southern South America includes Argentina, Brazil, Chile, Paraguay and Uruguay. Emerging data from Argentina, Chile and Uruguay revealed some coincidences between these countries, but also several differences, probably, as a result of multiple introduction events.     

Ophiostomatoid fungi associated with root‐feeding bark beetles on Scots pine in Poland

Ophiostomatoid fungi are known to be associated with various species of bark beetles. However, information about fungal associates of root‐feeding bark beetles in Europe is still fragmentary. For this reason, the fungal associates of Hylastes ater, H. opacus and Hylurgus ligniperda on Pinus sylvestris were isolated and identified. A total of 743 fungal isolates were collected and separated into 10 morphological groups. Analyses of ITS rDNA and partial β‐tubulin gene sequences confirmed that these groups represented distinct species. The 10 species included a total of 13 associations between fungi and bark beetles that had not been recorded previously. All of the bark beetles examined were frequently associated with ophiostomatoid fungi. The fungal diversity and relative abundance of species were very similar in the three species of root‐feeding bark beetles. The most commonly encountered associates of these beetles were Grosmannia radiaticola, Leptographium lundbergii, L. procerum and L. truncatum. Insect infestation data furthermore suggest that Hylastes spp. and Hg. ligniperda are also important vectors of the fungal pathogen Sphaeropsis sapinea.