Diversity of ophiostomatoid fungi associated with the large pine weevil, Hylobius abietis, and infested Scots pine seedlings in Poland

Context

Bark beetles are known to be associated with fungi, especially the ophiostomatoid fungi. However, very little is known about role of pine weevils, e.g., Hylobius abietis, as a vector of these fungi in Europe.

Aims

The aims of our study were to demonstrate the effectiveness of H. abietis as a vector of ophiostomatoid fungi in Poland and to identify these fungi in Scots pine seedlings damaged by weevil maturation feeding.

Methods

Insects and damaged Scots pine seedlings were collected from 21 reforestation sites in Poland. The fungi were identified based on morphology, DNA sequence comparisons for two gene regions (ITS, β-tubulin) and phylogenetic analyses.

Results

Sixteen of the ophiostomatoid species were isolated and identified. In all insect populations, Leptographium procerum was the most commonly isolated fungus (84 %). Ophiostoma quercus was also found at a relatively high frequency (16 %). Other ophiostomatoid fungi were found only rarely. Among these rarer fungi, four species, Leptographium lundbergii, Ophiostoma floccosum, Ophiostoma piliferum and Sporothrix inflata, were isolated above 3 %. L. procerum was isolated most frequently and was found in 88 % of the damaged seedlings. S. inflata was isolated from 26 %, while O. quercus occurred in 10 % of the seedlings.

Conclusion

This study confirmed that L. procerum and O. quercus were common associates of H. abietis, while others species were found inconsistently and in low numbers, indicating causal associations. H. abietis also acted as an effective vector transmitting ophiostomatoid species, especially L. procerum and S. inflata, to Scots pine seedlings.     

Diversity of Ophiostomatales species associated with conifer-infesting beetles in the Western Carpathians

Bark beetles (Coleoptera: Curculionidae, Scolytinae) are commonly recognised as important agents of tree mortality in coniferous forests of the Western Carpathians. They, together with weevils, are consistently associated with ophiostomatoid fungi. Information regarding conifer beetle-associated fungi in the Western Carpathians remains incomplete and unreliable, particularly with respect to fir-infesting bark beetles. This study aims to clarify associations between fungi in the genera Graphilbum, Leptographium, Ophiostoma and Sporothrix (Ophiostomatales) and their beetle vectors in Norway spruce (Picea abies), European larch (Larix decidua) and silver fir (Abies alba). Samples associated with 20 bark beetle species and weevils were collected from nine stands in Poland and the Czech Republic. Fungi were isolated from adult beetles and galleries. Isolates were identified based on morphology, DNA sequence comparisons for four gene regions (ITS, LSU, ß-tubulin, TEF 1-α) and phylogenetic analyses. In total, 46 distinct taxa were identified, including 25 known and 21 currently unknown species. Several associations between fungi and subcortical insects were recorded for the first time. In addition, O. borealis and O. quercus were detected from A. alba for the first time. The composition of the fungal communities varied among the studied tree species and to a lesser degree among the beetle species. The spruce-infesting bark beetles were commonly associated with species of Leptographium s. l. and Ophiostoma s. str.; the larch-infesting bark beetles were often associated with Ophiostoma s. str. and Sporothrix, while the fir-infesting bark beetles were commonly associated with Ophiostoma s. str. and Graphilbum. The most commonly encountered fungal associates of the examined insects were (a) Grosmannia cucullata, G. piceiperda, Grosmannia sp. 1, Ophiostoma macroclavatum and O. piceae with the spruce-infesting bark beetles; (b) O. pseudocatenulatum and Sporothrix sp. 1 with the larch-infesting bark beetles; and (c) O. piceae, Ophiostoma sp. 2 and Graphilbum sp. 2 with the fir-infesting bark beetles. The differences in fungal associates among the bark beetle species occurring on P. abies, L. decidua and A. alba could be linked to the different habitats that these beetles occupy.     

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.     

Ophiostomatoid fungi associated with three spruce-infesting bark beetles in Slovenia

Context

Ophiostomatoid fungi can severely affect the health and economic value of Norway spruce trees (Picea abies). Although the diversity of ophiostomatoid species and their associations with insects have been well-investigated in central and northern Europe, little is known about the conditions in south-eastern Europe.

Aim

This study aims to study the assemblages of ophiostomatoid fungi associated with three bark beetle species (Ips typographus, Ips amitinus, and Pityogenes chalcographus) that infect Norway spruce in Slovenia.

Methods

Bark beetles were sampled in four phytogeographic regions in Slovenia. The fungi found on the bark beetles were identified based on morphology, DNA sequence comparisons of ITS regions and phylogenetic analysis. The species compositions of the fungal associates of the three insect species were compared and the pairwise associations of the occurrence of the fungal species were analysed.

Results

Thirteen different species were found. The most commonly encountered fungal associates of the beetles were Ophiostoma bicolor, Ophiostoma brunneo-ciliatum, Grosmannia piceiperda, Ophiostoma ainoae, Ceratocystiopsis minuta, and Grosmannia penicillata. The composition of the fungal associates differed among the bark beetle species, but not among the phytogeographic regions.

Conclusions

This study confirms that ophiostomatoid species are common associates of the investigated bark beetle species. Many ophiostomatoid species have strong host associations. I. typographus and P. chalcographus can act as effective vectors for O. bicolor, O. ainoae, G. piceiperda and O. brunneo-ciliatum, whereas I. amitinus often carries G. piceiperda and C. minuta in Slovenian forests.     

The ability of the pine-top weevil to carry spores and infect Scots pine with Endocronartium pini

The pine-top weevil Pissodes piniphilus is known to be often associated with trees infected with resin-top disease. In this paper, the ability of the weevil to carry Endocronartium pini spores in laboratory conditions and its ability to infect pines in the field were studied. The weevil may carry spores and cause infection in healthy pines.     

Weevil feeding on Scots pine affects germination of Endocronartium pini

The aim of this study was to assess the effect and importance of the feeding of the pine top weevil (Pissodes piniphilus) on the germination of Endocronartium pini (syn. Peridermium pini) in Scots pine (Pinus sylvestris), and thus to establish the possibility of the E. pini infection via insect woundings. Germination tests were therefore carried out on current and previous year needle and phloem extracts. Elucidation of the importance of pathogen infection for the insect's feeding preference was also required; for this purpose feeding preference tests were carried out with healthy and infected pine branches using the pine top weevil as a test insect. Weevil feeding increased the germination of E. pini aeciospores on pine extracts. Germination on previous year annual-shoot extracts was lower than that on current year annual-shoot extracts. The advance of the growing season increased this trend, but weevil feeding increased germination on extracts from older annual shoots to the levels found on extracts from current annual shoots. Spores germinated equally well on needle extracts and on phloem extracts. The weevils ate more often on infected branches than on healthy branches. E. pini infections may occur via woundings on branches and weevil feeding may facilitate this.     

Effectiveness of modified White's solution at removing ascomycetes associated with the bark beetle Ips pini

Modified White's solution (1 g HgCl₂/l H₂O) is widely used to surface disinfest bark beetles of their phoretic fungi. We investigated the effectiveness of this solution at disinfesting adult Ips pini from its associated ophiostomatoid fungi. A treatment for 1, 4 or 8 min does not completely rid beetles of phoretic fungi, but does substantially reduce the amount of fungi they carry externally. Sterilizing with modified White's solution caused limited mortality (<16%).     

Association of the pine-top weevil with Endocronartium pini on Scots pine

It has been suggested that the pine-top weevil (Pissodes piniphilus) could be a vector of resin-top disease caused by Endocronartium pini. The association of this weevil with trees and stands infected with the fungus was studied. The number of pupal chambers around cankers and in trapping bolts indicated an association. Mobility studies with flight traps and sticky rings, however, gave inconclusive results as to the possible role of the weevil as vector of the disease.     

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.     

Associations between Pityogenes bidentatus and fungi in young managed Scots pine stands in Poland

The association between Pityogenes bidentatus and fungi was studied in young, managed Pinus sylvestris stands in Poland. Fungi were isolated from emerged adults and their galleries collected from four populations. In total, 2089 fungal isolates including 42 species, were obtained. Penicillium sp. 1 and Geosmithia sp. 1 were the most commonly isolated fungi from beetles (49% and 41% of beetles respectively). Geosmithia sp. 1 species was the dominant species in P. bidentatus galleries with a frequency of occurrence of 57.9%. Hormonema dematioides was the second most abundant fungus in gallery systems (17.1% of wood samples). Two of the isolated Geosmithia species were previously undescribed. Pityogenes bidentatus also vectored three ophiostomatoid species: Ophiostoma minus, O. piceae and Graphium sp. ‘W’. These species were occasionally isolated from beetles and their galleries, suggesting a non-specific relationship.     

Susceptibility of longleaf pine roots to infection and damage by four root-inhabiting ophiostomatoid fungi

Restoration of longleaf pine-dominated uplands is common on many public and private lands throughout the southeastern United States. The once dominant longleaf pine ecosystem is important to many now-threatened and endangered plant and animal species, and land managers are increasing efforts to reestablish this fire-dependent forest. Unfortunately, tree mortality in longleaf pine has been observed following attempts to re-introduce prescribed fire. Root-inhabiting ophiostomatoid fungi and their insect vectors have invaded roots of symptomatic longleaf pine. Although, the relationship between ophiostomatoid fungi and longleaf pine roots is poorly understood. In order to assess the pathogenicity and virulence of four ophiostomatoid fungi to longleaf pine, trees within two broad age classes (20–30 and 40–60 years) were used for root inoculations during the fall of 2006 and 2007 along with the spring of 2007 and 2008. All fungal species consistently caused resin-filled, discolored lesions on the phloem surface extending to the xylem. The successful inoculation of healthy longleaf pine roots confirms the pathogenicity of Grosmannia huntii, Leptographium procerum, Leptographium serpens, and Leptographium terebrantis. G. huntii caused the largest lesions, including 22.20 cm², 13.37 cm², and 9.21 cm² larger than L. procerum, L. terebrantis, and L. serpens respectively. In contrast, L. procerum caused significantly smaller lesions than all other fungi, including 8.65 cm² smaller than L. terebrantis and 10.69 cm² smaller than L. serpens. Restoration efforts of longleaf pine may be affected by fungal root infection in the future. Future studies should focus on the interactions between stress factors associated with longleaf pine to define more clearly the ecological role of root-inhabiting ophiostomatoid fungi in the ecosystem.     

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.     

Ophiostomatoid fungi associated with bark beetles onAbies veitchii in wave-regenerated forests

Relationships between tree mortality and bark beetle infestation onAbies veitchii at the wave-regenerated forest in Mt. Asahi, Okuchichibu area were investigated. Most of the firs with green needles and newly developed current year’s shoots in the dieback zone were heavily infested by bark beetles before the death of the trees. After heavy infestation of beetles, about half of the infested firs died within the year, and the other half died in the next year. When the species composition of bark beetles and associated ophiostomatoid fungi were investigated in Mt. Asahi and also at a typical wave-regenerated forest in Mt. Shimagare, Yatsugatake area,Cryphalus montanus andC. piceae were dominant beetle species for Mt. Asahi and Mt. Shimagare, respectively.Ophiostoma subalpinum andO. europhioides were dominant fungal species at both wave-generated forests. BecauseO. subalpinum was detected more frequently from deeper areas of sapwood thanO. europhioides, it was suggested that the fungal species may accelerate the death of stressed firs in wave-regenerated forests. Contribution No. 169, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba.     

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.     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

A survey of Ophiostoma species vectored by Hylastes ater to pine seedlings in New Zealand

Ecological studies in New Zealand require consideration of the impact of exotic (introduced) organisms from disparate environments. A survey of Ophiostoma species isolated and identified from Pinus radiata stumps, the bark beetle Hylastes ater collected from these stumps and from P. radiata seedlings following sublethal damage by H. ater, was undertaken in 10 reforestation sites in New Zealand. Nine Ophiostoma species were isolated and identified in this survey. Ophiostoma galeiformis and Ophiostoma huntii were isolated most frequently from all the substrates sampled. Ophiostoma floccosum, Ophiostoma setosum, Leptographium procerum and Leptographium truncatum were also isolated from stumps, damaged seedlings and beetles. Ophiostoma quercus was only isolated from stumps and beetles and Ophiostoma ips only from beetles. A strong relationship between the severity of H. ater feeding damage, and the presence and number of species of Ophiostoma was found. Eight Ophiostoma species were transmitted by H. ater to P. radiata seedlings in laboratory transmission experiments. From the results of this field survey and the laboratory experiments we confirm that H. ater is a vector of Ophiostoma species to P. radiata seedlings in New Zealand.     

First record of Ektaphelenchoides pini associated with Ips sexdentatus on Pinus nigra laricio in Italy

In February 2015, an unexpected windstorm downed five hectares of a European black pine Pinus nigra subsp. laricio forest formation located close to Vallombrosa, Florence (Central Italy). In the following spring, an extensive survey was conducted in the area. Felled trees, stumps and all the suitable plant material were screened for the presence of the pinewood nematode (PWN), Bursaphelenchus xylophilus, by sampling wood and bark. Bark beetles were then collected from the gallery systems on the inner side of bark samples and observed in the laboratory. The following bark beetles were morphologically identified: Ips sexdentatus, Orthotomicus erosus, O. laricis and Pityogenes bidentatusa. The dissection of Ips sexdentatus allowed the extraction of numerous nematodes that were morphologically and molecularly identified as Ektaphelenchoides pini. Conversely, only few nematode specimens were isolated from either pine bark or wood. These individuals could be only molecularly identified and belonged to an undescribed nematode taxon. Even though no PWN was recorded in the investigated sites, our survey allowed the detection of a new association between E. pini and I. sexdentatus on P. nigra.     

Larval cannibalism and fitness in the stored grain weevils <Emphasis Type="Italic">Sitophilus granarius</Emphasis> and <Emphasis Type="Italic">Sitophilus zeamais</Emphasis>

Resource limitation is an important determinant of life history and behavior while mediating competition and reproduction among organisms. Discreet and closed systems such as grain kernels and seeds impose drastic restrictions to grain beetles that spend their immature stages within a single kernel selected by their mother. This is the case of internally feeding stored grain beetles, such as the grain weevils. Female egg-laying decisions and larval competition largely determine resource limitation for such insects where clustered egg distribution and contest competition with larval interference and cannibalism take place. As the clustered eggs within a grain lead to larval competition and conspecific weevil larvae face each other off during development allowing the emergence of one or two larvae per kernel, we hypothesized that such competition and consequent cannibalism will have fitness consequences for the competing individuals and their offspring. Thus, larvae of the granary weevil (Sitophilus granarius L.) and the maize weevil (Sitophilus zeamais Motsch.) were subjected to larval competition with cannibalism, and lack of it, to assess the potential fitness consequence of cannibalism on these non-carnivorous pest species of stored grains. Larval cannibalism reduced developmental time of maize weevil, but not of granary weevil. However, such condition led to heavier adult weevils of both species exhibiting higher reproductive output generating more and better quality progeny than non-cannibal weevils. These findings indicate direct nutritional benefits of cannibalism to grain weevils favoring their status of key pest species of stored cereal grains.     

Beetles (Coleoptera) caught with pheromones of Gnathotrichus retusus and G. sulcatus (Col., Scolytidae) in southern Finland

The ambrosia beetle Gnathotrichus materiarius, which originally came from North America, was discovered in southern Finland in 1996. In 1997, using Norwegian drainpipe traps baited with pheromones of Gnathotrichus retusus and G. sulcatus, we collected beetles in the region where the first specimen had been caught in order to determine whether this potential pest species had become established in the area. Samples from a total of 16 traps included 79 species of beetles and 719 individuals, but no specimens of G. materiarius. The most abundant species in the samples were the ambrosia beetles Xyleborus dispar and Trypodendron lineatum. Several predators and other associates of bark beetles were also captured. The majority of the beetles caught were saproxylic species.     

The virulence of phytopathogenic fungi associated with the bark beetles Tomicus piniperda and Orthotomicus erosus in Tunisia

Phytopathogenic fungi associated with the bark beetles Tomicus piniperda and Orthotomicus erosus were isolated in various pine forests of Tunisia. Tomicus piniperda and its galleries yielded Leptographium wingfieldii, Ophiostoma minus, and Ophiostoma ips. Ophiostoma minus was the most frequent species associated with T. piniperda, in both the attacking and the emerging beetles. It was collected from most investigated forests, whereas O. ips and L. wingfieldii were obtained only from forests located in Central and Northern Tunisia. Frequencies of association with T. piniperda were always low, reaching 11.1% only once, for O. ips. Ophiostoma ips was the only blue stain fungus associated with O. erosus and its galleries. It was found in all the localities, but at a low and variable frequency, exceeding 15% very rarely. The virulence of 16 fungal isolates was tested by single inoculations into Pinus halepensis (Aleppo pine) at two localities. Differences were detected among species; L. wingfieldii was the most virulent and O. minus the least virulent species in terms of phloem reaction zone formation and fungal growth in the phloem. In a separate experiment, mass inoculations (400 and 800 inoculations per m² of bole on 1‐m high belts) were performed with two isolates of L. wingfieldii on Aleppo pine and on Pinus brutia (Brutia pine). Three months later, measurements of sapwood status (% of conductive transversal section) and of its specific hydraulic conductivity, as well as of the phloem reaction zone lengths, did not show any isolate or density effect. At these experimental sites, Brutia pines appeared significantly more susceptible than Aleppo pines.