A survey of microbial antagonists of Agriotes wireworms from Germany and Italy

Wireworms of the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests affecting numerous crops throughout the world and causing damage of economic importance. Between 2008 and 2011, a survey of the occurrence of natural microbial antagonists of wireworms was conducted. Altogether, 3,420 wireworms and two dead beetles from agricultural habitats and grassland of various parts of Germany and 700 lab-reared wireworms from Italy were screened for the presence of disease. Of these, 283 selected dead and moribund Agriotes specimens were diagnosed by light and electron microscopy for insect pathogens; 66 wireworms contained unspecified bacterial infections; and 86 specimens displayed fungal growth with 25 infected by the entomopathogenic taxa Beauveria bassiana, Metarhizium anisopliae, Isaria fumosorosea, Isaria spp. and Lecanicillium sp. Nematodes were observed in 29 dead individuals, and 141 individuals showed no distinguishable pathogens. In one dead Agriotes larva, an intracellular bacterium was identified as a new Rickettsiella pathotype named ‘Rickettsiella agriotidis’. The survey resulted in a natural infection rate of 0.66 %. The possible use of these pathogens as biocontrol agents is discussed.     

Effect of aging in adult Monochamus alternatus (Coleoptera: Cerambycidae) on the susceptibility of the beetle to Beauveria bassiana (Ascomycota: Hypocreales)

To clarify the effect of aging in adult Monochamus alternatus on the susceptibility of the beetle to Beauveria bassiana, we inoculated the fungus into adult beetles 0, 3, 7, 14, and 28 days after emergence. The number of days from B. bassiana inoculation to M. alternatus death increased with the number of days from beetle emergence to inoculation up to 14 days after emergence. This indicated that the susceptibility of adult beetles to the fungus decreased with aging. However, B. bassiana-inoculated beetles 28 days after emergence died earlier after inoculation than those inoculated with the fungus 14 days after emergence; i.e., the beetle’s susceptibility to the fungus increased again. We considered this phenomenon to be the result of senescence of adult beetles. We concluded that the susceptibility of M. alternatus to B. bassiana decreased first and then increased again with aging.     

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.     

Occurrence of Agriotes wireworms in Austrian agricultural land

Agriotes wireworms (Coleoptera: Elateridae) are abundant soil-dwelling herbivores which can inflict considerable damage to field crops. In Europe up to 40 species occur, differing in their ecology and pest status. Their distribution in the larval stage, however, has rarely been assessed because of the considerable effort in collecting wireworms and the difficulties in identifying them to species-level. Here, we examined the occurrence of Agriotes wireworms in Austrian agricultural land with regard to their association with climatic and soil parameters. Using a molecular identification system, 1348 field-collected larvae from 85 sites were identified to species-level. Three species, Agriotes obscurus, Agriotes brevis, Agriotes ustulatus, and two that could not be discerned molecularly (Agriotes lineatus and Agriotes proximus), were assigned to two ecological groups: (i) A. brevis/A. ustulatus, found in areas with a warmer, drier climate and alkaline soils, and (ii) A. obscurus/A. lineatus/proximus which occur mainly at higher altitude characterised by lower temperatures, higher precipitation and acidic, humus-rich soils. Agriotes sputator was abundant throughout Austria, confirming its euryoecious nature. Only one larva of Agriotes litigiosus was found, prohibiting further analysis. These data contribute to a characterisation of species-specific traits in Agriotes larvae in agricultural land, an important prerequisite to develop efficient control strategies for these wireworms.     

The Asian‐Melanesian bambusicolous genus Myriodiscus is related to the genus Tympanis,the North American‐European tree pathogen

Species of Tympanis are well‐known pathogens in Holarctic forests, but we know little about their relationship to other genera in Tympanidaceae. The genus Myriodiscus, remarkable macroscopically and a possible pathogen on bamboo, has a complicated taxonomic history and has not been conclusively placed phylogenetically. Species of Myriodiscus have been described under two other generic names, Ascotremellopsis and Gelatinomyces, the latter being related to the pathogenic genus Collophorina. There has been no formal synonymy of these three bambusicolous genera or agreement on their systematic positions. We combine accurate micromorphology and DNA sequence data to show the link between Tympanis and Myriodiscus and reveal previously unrecognized features of the latter. These two genera show a type of ascus development unique in the Leotiomycetes. With this new data, we have resolved past confusions over the identity of these fungi, determined their systematic position and have proposed the proper synonymies for Myriodiscus sparassoides and one new combination (M. conus).     

Genetic variability of Alder yellows phytoplasma in Alnus glutinosa in its natural Spreewald habitat

The genus ‘Candidatus Phytoplasma’ comprises wall‐less bacteria colonizing the phloem of plants and insect tissues. Phytoplasmas are associated with diseases in over 1000 plant species worldwide, including many important crops as well as forest trees. Alder yellows (AldY) phytoplasma, which frequently infects Alnus spp., is closely related to the economically important phytoplasma causing Flavescence dorée (FD) in grapevines. In a natural habitat (Spreewald, Brandenburg, Germany), 57 Alnus glutinosa (black alder) trees were examined for phytoplasma infection in summer 2013. No phytoplasma typical infection‐associated symptoms such as yellowing and decline were observable in this natural swamp‐alder area. Amplification followed by a restriction fragment length polymorphism, and a sequence analysis of the 16S rDNA, allowed for the detection of AldY phytoplasmas in all examined trees and their assignment to the taxonomic group 16SrV‐C. Additional analyses of the non‐ribosomal marker gene methionine aminopeptidase (map) revealed diverse strains as well as mixed infections with closely related AldY strains, and the strains were assigned to phylogenetic clusters closely related to German Palatinate grapevine yellows, AldY or FD strains. The results confirmed that AldY phytoplasmas infection in A. glutinosa is prevalent. The results also indicate a presence of an established phytoplasma population in chronically infected black alder.     

Joint action of Beauveria bassiana and the insect growth regulators diflubenzuron and novaluron, on the migratory locust, Locusta migratoria

Beauveria bassiana (Balsamo) Vuillemin and sublethal concentrations of the insect growth regulators (IGR) diflubenzuron and novaluron were applied simultaneously and sequentially to second-instar Locusta migratoria migratorioides (Sauss.) to determine the interaction between these materials and an entomopathogenic fungus. Nymphs were fed on corn leaf disks treated with several concentrations of each IGR, and a constant dose of B. bassiana was applied topically. Additive interaction was demonstrated in all instances when second-instar nymphs were exposed to diflubenzuron or novaluron simultaneously with B. bassiana treatment, and when the fungus was applied first and IGR after 48 h. Additive interaction was also exhibited when novaluron was applied first and B. bassiana after 48 h. Antagonism was demonstrated when nymphs were fed diflubenzuron-treated corn leaves first, then after 48 h were treated with B. bassiana. The additive interaction that was observed in our experiments could still be useful to achieve efficacious levels of acridid control at low rates of an IGR along with a biological control agent such as an entomopathogenic fungus.     

Honeybush (Cyclopia sp.) – A rich source of compounds with high antimutagenic properties

The genus Cyclopia (Fabaceae family) includes a number of shrubs endemic to Cape Fynbos region of South Africa. The most common of these plants is Cyclopia intermedia E. Mey., used together with other Cyclopia species (mainly C. subternata and C. sessiliflora) to manufacture the honeybush herbal tea which has been produced in South Africa roughly since the beginning of the 19th century. Honeybush infusions are gaining popularity due to their characteristic honey-like flavour, low tannin content, absence of caffeine and potential health effects related to their antimutagenic and antioxidant properties.     

Transformation and attachment of Beauveria bassiana conidia on the cuticle of Tribolium confusum and Sitophilus oryzae in conjunction with diatomaceous earth

Agrobacterium tumefaciens-mediated transformation (AMT) was used in order to create fluorescent conidia of Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales). From the fungal transformants, one Enhanced Green Fluorescent Protein B. bassiana strain indicated stability (maintained the GFP characteristics) in fluorescent signal, while its pathogenicity did not differ significantly from the wild B. bassiana type. After exposure on wheat treated with the transformed conidia, fluorescence was evident on all body parts but especially on metasternon of adults of Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) and on elytra in of Sitophilus oryzae (L.) (Coleoptera: Curculionidae). No significant differences were noted in fluorescence levels in the case of T. confusum between the transformed and the wild strain. However, the addition of diatomaceous earth (DE) increased attachment, especially at increased intervals after exposure. For S. oryzae, fluorescence of the transformed strain was decreased with the increased post-exposure time, but the simultaneous presence of DE increased fluorescence. The results of this study demonstrate that fluorescent conidia of B. bassiana can be utilized as a tool for the evaluation of parameters that affect the attachment of entomopathogenic fungi in insects?? cuticle.     

The susceptibility of Asian,European and North American <Emphasis Type="Italic">Fraxinus</Emphasis> species to the ash dieback pathogen <Emphasis Type="Italic">Hymenoscyphus fraxineus</Emphasis> reflects their phylogenetic history

In Europe, common ash (Fraxinus excelsior) is being decimated because of the invasive fungus Hymenoscyphus fraxineus. In its native range in Asia this ascomycete is considered a harmless leaf associate of F. mandshurica and F. chinensis subsp. rhynchophylla. Field observations from Europe suggest that there is species-specific variation in disease susceptibility among European and North American Fraxinus species, but a wider comparison at the genus level has been missing so far. We assessed disease symptoms and pathogen apothecium development in 17 Fraxinus species from Asia, Europe and North America exposed to high infection pressure in a Danish arboretum. We also tested their susceptibility to pathogen infection through controlled stem and leaf inoculations and subsequently assessed the level of pathogen DNA by a qPCR assay. The results suggested the presence of a phylogenetic signal in disease susceptibility where closely related Asian, European and North American species in section Fraxinus had relatively high levels of H. fraxineus DNA in the leaves and supported high production of apothecia. Leaves from some North American species also contained relatively high levels of H. fraxineus DNA, supported moderate production of apothecia and developed lesions—stating the need to avoid introduction of H. fraxineus to North America.     

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.     

Distribution of adult stages of soil insect pests across an agricultural landscape

The landscape scale distributions of adult male click beetles of the species Agriotes lineatus, A. obscurus and A. sputator, and the cranefly Tipula paludosa were studied using traps along transects over 2 years. The transects (0.7–1.7 km with either sex pheromone (Agriotes) or water (T. paludosa) traps at 100 m spacings) were in an area of mixed organic land use. Agriotes lineatus was the most numerous click beetle caught even though its larvae were absent from these fields. Greater trap counts were found for all taxa except A. obscurus in grassland. The number of click beetles were influenced by the sampling year, crop type and the species, and interactions of other variables, suggesting that sex pheromone trap counts exhibit variability because of the complexity of the environment. Trap catches were spatially clustered with the exception of A. lineatus in 2005 which had a random distribution. Only A. sputator showed a significant, positive relationship between counts in 2005 and 2006. Variation in catch count of crane flies was largely determined by crop type. We conclude that Agriotes spp. disperse widely and may be recruited from outside the field which makes the interpretation of sex pheromone trap counts scientifically challenging.     

Effects of landscape composition and configuration on northern flying squirrels in a forest mosaic

Habitat loss and sometimes habitat fragmentation per se affect species survival, reproduction, dispersal, abundance and distribution. However, understanding the independent effects of fragmentation (i.e., landscape configuration) has been limited because it is frequently confounded with landscape composition (i.e., habitat amount). We assess the independent effects of landscape composition and configuration on the occurrence of northern flying squirrels (Glaucomys sabrinus) in New Brunswick, Canada after controlling for local site conditions. We measured landscape structure using an “organism-based” approach; landscape structure was characterized quantitatively using a spatially explicit local-scale distribution model for northern flying squirrels.     

Phylogeographic variation among isolates of the Sirococcus conigenus P group

In this study the phylogeographic variation among isolates of the Sirococcus conigenus P group and the phylogenetic relationships of S. conigenus with Sirococcus clavigignenti‐juglandacearum and other species previously placed in the genus Sirococcus were investigated. A collection of 33 isolates originating from Picea, Pinus and Larix in Europe, North America and Bhutan were characterized by sequence analyses of the internal transcribed spacer (ITS) region (including ITS1, 5.8S ribosomal DNA, ITS2) of the nuclear rDNA and a portion of the β‐tubulin gene. In phylogenetic analyses most isolates from pine, spruce and larch formed a distinct clade, representing the P group of S. conigenus, which was separated from the T group of this pathogen. Four isolates from Picea in Europe and Canada formed a third clade within S. conigenus and these isolates are referred to as the S group. The P group consisted of five distinct ITS haplotypes, which partly differed in their optimum growth temperature and their growth rates at 25°C on malt extract agar. Nested clade analysis resolved the five haplotypes into three distinct clades and revealed significant genetic/geographic associations for some of the haplotypes. Parsimony analysis of the small subunit (18S) ribosomal DNA sequences confirmed the phylogenetic affinities between S. conigenus and S. clavigignenti‐juglandacearum. In contrast, Godronia cassandrae and Hormococcus conorum, which formerly had been placed in the genus Sirococcus, were found to be only distantly related to S. conigenus and S. clavigignenti‐juglandacearum.     

Potential of Metarhizium anisopliae and Beauveria bassiana in the control of tea termite Microtermes obesi Holmgren in vitro and under field conditions

Two strains of Metarhizium anisopliae (Metschnikoff) Sorokin and two strains of Beauveria bassiana (Ballsamo) Vuillemin were evaluated for their potential use as biological control agents against the tea termite Microtermes obesi Holmgren. The strains were screened for relative pathogenicity, and the median lethal time eliciting 50% mortality (LT₅₀) was calculated. In general, the M. anisopliae strains were more virulent with lower LT₅₀ values than B. bassiana strains. The LT₅₀ values ranged from 1.6 to 3.7 days. The median lethal concentrations (LC₅₀) of all the isolates were also determined. The M. anisopliae strains had low LC₅₀ values compared to B. bassiana strains. The LC₅₀ values ranged from 35 to 140 conidia per termite. The strains were also tested for survival under laboratory nest conditions. All the isolates exhibited the ability to grow, sporulate and produce an epizootic in treated nest material. Field applications of the isolates also produced promising results.     

Effects of insect activity, soil, and cuticular factors on virulence of Beauveria bassiana toward Blissus leucopterus hirtus

The fungus Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) is a potential biological control agent of hairy chinch bug, Blissus leucopterus hirtus Montandon (Hemiptera: Lygaeidae), but its virulence against this important turf grass pest is limited. To gain insight into why high numbers of conidia are needed for rapid kill, conidial persistence on insects was monitored under several experimental conditions. One day after exposure to a concentrated B. bassiana suspension, most conidia were removed from insects, particularly at warmer temperatures. Removal was higher when soil was present, suggesting that the insects may have transferred the conidia to soil. Most conidia on insects lost viability within 2?days. In contrast, conidia stored in soil under the same conditions as the insects remained viable. Earlier instar nymphs were more susceptible to lethal fungal infection than were adults. Materials recovered from a hexane rinse of adult insects inhibited conidial germination. These results have implications for applied aspects of chinch bug biological control with B. bassiana.     

Zur Verwendbarkeit von Fungiziden als experimentaltechnische Hilfsmittel bei Untersuchungen an Borkenkäfern

The fungicides, Euparen and Orthocid 50, proved to be efficient in suppressing growth of saprophagous fungi on spruce phloem chips, which are used as a food ofPityogenes chalcographus adults in laboratory experiments.Beauveria bassiana infection of the beetles is also checked, when fungicides have been applied to the food.     

银杏内生细菌XZNUM033的鉴定及其抗杨树变色真菌活性物质的理化性质

银杏内生细菌XZNUM 033对杨树变色真菌—可可球二孢菌具有很强的抑制活性,通过对该菌株培养液一些理化性质的测定,结果表明:培养液在pH值为7.0时,其抗可可球二孢菌活性最强,培养液对温度、光照、紫外线都具有较好的稳定性。根据银杏内生细菌XZNUM 033的菌落、菌体形态、革兰氏染色、芽孢染色、生理生化特性以及16S rRNA序列分析,初步把银杏内生细菌XZNUM 033定为解淀粉芽孢杆菌。     

Geographic distribution of chestnut feeding insects in Greece

Greece has been traditionally considered as a chestnut-producing country; yet, chestnut production has experienced a dramatic decline in the last decades, mainly due to chestnut blight Cryphonectria parasitica and chestnut feeding pests. Though chestnut blight is partially under control, little attention has been granted on chestnut feeding pests in Greece. Two species are described as major pests, i.e., Cydia splendana and Curculio elephas, without any information regarding their distribution. This study aimed to identify species diversity that feed on chestnuts trees using genetic markers and further to obtain insight into their distribution. Besides C. splendana and C. elephas, three additional species were determined namely Cydia fagiglandana, Curculio glandium as well as a Curculio sp. that has never been genetically determined before. The distribution of these species was inhomogeneous in Greece. These results demonstrate clearly the need for a carefully planned pest management approach adapted to the local mixture of chestnut feeding pests of each area rather than one general approach.