Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome‐wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.     

Exploring the role of asexual multiplication in poplar rust epidemics: impact on diversity and genetic structure

Fungal plant pathogens, especially rust fungi (Pucciniales), are well known for their complex life cycles, which include phases of sexual and asexual reproduction. The effect of asexual multiplication on population genetic diversity has been investigated in the poplar rust fungus Melampsora larici‐populina using a nested hierarchical sampling scheme. Four hierarchical levels were considered: leaf, twig, tree and site. Both cultivated and wild poplar stands were sampled at two time points at the start and end of rust epidemics. A total of 641 fungal isolates was analysed using nine microsatellite markers. This study revealed that the genetic signature of asexual multiplication in the wild poplar stand was seen only at lower hierarchical levels (leaf and twig). Moreover, we observed an erosion of clonal structure through time, with an increase in both gene and genotypic diversity. New genotypes contributed to host infection over time, which demonstrates the importance of allo‐infection in the epidemic process in this host‐pathogen system. Compared with the wild stands, the nearly lack of detection of clonal structure in the cultivated stands reflects the higher infection level on cultivated poplars. More generally, this genetic analysis illustrates the utility of population genetics approach for elucidating the proportion of asexual reproduction in the multiplication of isolates during an epidemic, and for proper quantification of asexual dispersal in plant pathogens.     

Insect pests of Norway spruce cones: incidences and altitude preferences with emphasis on Cydia strobilella (L.)

Pest species infesting spruce cones were identified from 109 locations approved for the collection of seed material in the Czech Republic. Four (occasionally five) cones were studied from each location, and 448 cones were examined in total. Each cone was assessed for external damage and was then cut open along the spine. The predominant pest species, Cydia strobilella, was detected in 65% of the cones, while the next most common species, Dioryctria abietella, was detected in 13% of the cones. Ernobius abietis, Eupithecia sp., and Thekopsora areolata were less numerous. Kaltenbachiola strobi and Megastigmus sp. were recorded sporadically. Most cones (68%) were infested with only one species. The maximum number of C. strobilella larvae detected in one cone was 13. Cone damage resulting from C. strobilella declined markedly with increasing elevation. Thus, cone infestation by C. strobilella was greater in those forest vegetation zones located in lowlands and uplands rather than in highlands and at lower mountain elevations. The number of cones without damage tended to increase with elevation, but infestation by D. abietella was unrelated to elevation. In some cases, C. strobilella damage to cones was severe. External markings on the cone (resin and deformation) cannot by themselves serve as reliable criteria for predicting C. strobilella infestation, although cones with such signs should be preferentially assessed when seed material is inspected.     

On the hunt for the alternate host of Hemileia vastatrix

Coffee leaf rust (CLR), caused by the fungal pathogen Hemileia vastatrix, has plagued coffee production worldwide for over 150 years. Hemileia vastatrix produces urediniospores, teliospores, and the sexual basidiospores. Infection of coffee by basidiospores of H. vastatrix has never been reported and thus far, no alternate host, capable of supporting an aecial stage in the disease cycle, has been found. Due to this, some argue that an alternate host of H. vastatrix does not exist. Yet, to date, the plant pathology community has been puzzled by the ability of H. vastatrix to overcome resistance in coffee cultivars despite the apparent lack of sexual reproduction and an aecidial stage. The purpose of this study was to introduce a new method to search for the alternate host(s) of H. vastatrix. To do this, we present the novel hypothetical alternate host ranking (HAHR) method and an automated text mining (ATM) procedure, utilizing comprehensive biogeographical botanical data from the designated sites of interests (Ethiopia, Kenya and Sri Lanka) and plant pathology insights. With the HAHR/ATM methods, we produced prioritized lists of potential alternate hosts plant of coffee leaf rust. This is a first attempt to seek out an alternate plant host of a pathogenic fungus in this manner. The HAHR method showed the highest‐ranking probable alternate host as Psychotria mahonii, Rubus apetalus, and Rhamnus prinoides. The cross‐referenced results by the two methods suggest that plant genera of interest are Croton, Euphorbia, and Rubus. The HAHR and ATM methods may also be applied to other plant–rust interactions that include an unknown alternate host or any other biological system, which rely on data mining of published data.     

Life cycle and life strategy features of Puccinia glechomatis (Uredinales) favorable for extending the natural range of distribution

The objective of this study was to find features in microcyclic rust fungi (Uredinales) on wild host plants favorable for extension of the natural range of distribution. Puccinia glechomatis, a leptosporic rust fungus and its herbal host Glechoma hederacea (Lamiaceae), both natives to Eurasia and introduced in North America, were used for this study. Although the host has been known from North America since the beginning of the nineteenth century, the rust fungus was first observed there only in recent years. Favorable features were identified by studying the life cycle of the rust, including nuclear conditions and seasonal characteristics as well as its spread in North America. The life cycle was studied macroscopically by inoculation experiments, by various light microscope techniques, and by scanning electron microscopy. The spread of the pathogen and its host were reconstructed by evaluating host plant herbarium specimens and databases, literature, and field study data. The studies on P. glechomatis show that, generally for microcyclic rust fungi, establishment and potential for spread are based on several favorable features of both the host (e.g., synanthropic occurence and dispersal, genetic stability, regeneration of vegetative plant parts) and the rust fungus (asexual reproduction/genetic stability, homothallism, propagation with host plant, formation of both leptospores and thick-walled teliospores).     

Beyond animals and plants: dynamic maternal effects in the fungus Neurospora crassa

Maternal effects are widely documented in animals and plants, but not in fungi or other eukaryotes. A principal cause of maternal effects is asymmetrical parental investment in a zygote, creating greater maternal vs. paternal influence on offspring phenotypes. Asymmetrical investments are not limited to animals and plants, but are also prevalent in fungi and groups including apicomplexans, dinoflagellates and red algae. Evidence suggesting maternal effects among fungi is sparse and anecdotal. In an experiment designed to test for maternal effects across sexual reproduction in the model fungus Neurospora crassa, we measured offspring phenotypes from crosses of all possible pairs of 22 individuals. Crosses encompassed reciprocals of 11 mating‐type ‘A’ and 11 mating‐type ‘a’ wild strains. After controlling for the genetic and geographic distances between strains in any individual cross, we found strong evidence for maternal control of perithecia (sporocarp) production, as well as maternal effects on spore numbers and spore germination. However, both parents exert equal influence on the percentage of spores that are pigmented and size of pigmented spores. We propose a model linking the stage‐specific presence or absence of maternal effects to cellular developmental processes: effects appear to be mediated primarily through the maternal cytoplasm, and, after spore cell walls form, maternal influence on spore development is limited. Maternal effects in fungi, thus far largely ignored, are likely to shape species' evolution and ecologies. Moreover, the association of anisogamy and maternal effects in a fungus suggests maternal effects may also influence the biology of other anisogamous eukaryotes.     

How to find a needle in a haystack – host plant finding of the weevil Ceratapion onopordi

The weevil Ceratapion onopordi Kirby (Coleoptera: Apionidae) shows a mutualistic interaction with the rust fungus Puccinia punctiformis (Str.) Röhl (Pucciniaceae). One of the weevil’s host plants, the thistle Cirsium arvense (L.) Scop. (Asteraceae), is also the host of the rust fungus. It has been argued that weevils prefer rust‐infected thistle shoots for egg deposition and consequently should be able to detect such shoots. Olfactory, visual, and gustatory orientation was tested using a four‐chamber olfactometer, a visual testing arena, and feeding choice tests. Whereas the weevils used olfactory cues to find their host plants, visual orientation does not seem to be important. Rust‐infected thistle shoots were not preferred over healthy shoots in any of the tests. We conclude that host plants infected with rust fungi, which are rather rare in the field, appear to be found more or less by chance.     

Endophyte communities vary in the needles of Norway spruce clones

Endophytic fungi show no symptoms of their presence but can influence the performance and vitality of host trees. The potential use of endophytes to indicate vitality has been previously realized, but a standard protocol has yet to be developed due to an incomplete understanding of the factors that regulate endophyte communities. Using a culture-free molecular approach, we examined the extent to which host genotype influences the abundance, species richness, and community composition of endophytic fungi in Norway spruce needles. Briefly, total DNA was extracted from the surface-sterilized needles of 30 clones grown in a nursery field and the copy number of the fungal internal transcribed spacer (ITS) region of ribosomal DNA was estimated by quantitative PCR. Fungal species richness and community composition were determined by denaturing gradient gel electrophoresis and DNA sequencing. We found that community structure and ITS copy number varied among spruce clones, whereas species richness did not. Host traits interacting with endophyte communities included needle surface area and the location of cuttings in the experimental area. Although Lophodermium piceae is considered the dominant needle endophyte of Norway spruce, we detected this species in only 33 % of samples. The most frequently observed fungus (66 %) was the potentially pathogenic Phoma herbarum. Interestingly, ITS copy number of endophytic fungi correlated negatively with the richness of ectomycorrhizal fungi and thus potential interactions between fungal communities and their influence on the host tree are discussed. Our results suggest that in addition to environmental factors, endophyte communities of spruce needles are determined by host tree identity and needle surface area.     

Microcycle conidiation — A review

Microcycle conidiation is defined as the germination of spores by the direct formation of conidia without the intervention of mycelial growth, as occurs in most normal life cycles. It is a method of asexual spore formation in which the normal life cycle of the fungus is bypassed. Spores formed through sexual reproduction and species with unicellular thalli are not included in microcycle conidiation. The term secondary conidium or secondary spore is usually, but not always, synonymous with microcycle conidiation. In the laboratory various factors, but especially temperature, can induce the microcycle condition in such fungi asAspergillus niger, Penicillium andNeurospora crassa, providing a useful tool for research. Microcycle conidiation has also been reported in a broad range of species in nature, and comprises a normal part of the life cycle in several groups, including the Entomophthorales, Taphrinales, Clavicipitales, Uredinales, Ustilaginales, Tremellales and Exobasidiales. The presence of a microcycle in such fungi undoubtedly provides a survival mechanisn for spores that encounter unfavorable conditions.     

Completion of the sexual cycle and demonstration of genetic recombination in Ustilago maydis in vitro

The heterobasidiomycetes responsible for plant smuts obligatorily require their hosts for the completion of the sexual cycle. Accordingly, the sexual cycle of these fungi could so far be studied only by infecting host plants. We have now induced Ustilago maydis, the causative agent of corn smut, to traverse the whole life cycle by growing mixtures of mating-compatible strains of the fungus on a porous membrane placed on top of embryogenic cell cultures of its host Zea mays. Under these conditions, mating, karyogamy and meiosis take place, and the fungus induces differentiation of the plant cells. These results suggest that embryogenic maize cells produce diffusible compounds needed for completion of the sexual cycle of U. maydis, as the plant does for the pathogen during infection. Received: 16 February 1999 / Accepted: 30 June 1999     

The diversity of nuclear cycle in microcyclic rust fungi (Uredinales) and its ecological and evolutionary implications

 Nine types with 11 variations of nuclear cycle and associated metabasidium development were distinguished in microcyclic rust fungi. An additional type was recognized in rust fungi with an expanded life cycle. A significant proportion of rust fungi with a reduced life cycle is assumed to have lost a sexual genetic recombination process, being either apomictic or asexual in reproduction. Most species that retain a sexual process in the microcyclic life cycle seem to have become homothallic. During life cycle evolution by the omission of spore stages, these traits might have had a selective advantage for those species that had less opportunity to encounter a genetically different but sexually compatible mate because of isolated patchy distribution or a short growing season. The findings that different populations of a morphologically identifiable species exhibit two or more distinct patterns of nuclear cycle and different metabasidium development indicate that microcyclic lineages might have evolved independently and repeatedly from a macrocyclic parental species. Those lineages are morphologically the same but would differ from each other in their genetics and biology. Received: July 5, 2002 / Accepted: August 5, 2002 Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research (no. 09640744) from the Ministry of Education, Science and Culture (now the Ministry of Education, Culture, Sports, Science and Technology), Japan. Correspondence to:Y. Ono     

TECHNICAL ADVANCE: Transformation of the flax rust fungus,Melampsora lini: selection via silencing of an avirulence gene

Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium‐mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non‐transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.     

Inferring the contribution of sexual reproduction,migration and off‐season survival to the temporal maintenance of microbial populations: a case study on the wheat fungal pathogen Puccinia striiformis f.sp. tritici

Understanding the mode of temporal maintenance of plant pathogens is an important domain of microbial ecology research. Due to the inconspicuous nature of microbes, their temporal maintenance cannot be studied directly through tracking individuals and their progeny. Here, we suggest a series of population genetic analyses on molecular marker variation in temporally spaced samples to infer about the relative contribution of sexual reproduction, off‐season survival and migration to the temporal maintenance of pathogen populations. We used the proposed approach to investigate the temporal maintenance of wheat yellow rust pathogen, Puccinia striiformis f.sp. tritici (PST), in the Himalayan region of Pakistan. Multilocus microsatellite genotyping of PST isolates revealed high genotypic diversity and recombinant population structure across all locations, confirming the existence of sexual reproduction in this region. The genotypes were assigned to four genetic groups, revealing a clear differentiation between zones with and without Berberis spp., the alternate host of PST, with an additional subdivision within the Berberis zone. The lack of any differentiation between samples across two sampling years, and the very infrequent resampling of multilocus genotypes over years at a given location was consistent with limited over‐year clonal survival, and a limited genetic drift. The off‐season oversummering population in the Berberis zone, likely to be maintained locally, served as a source of migrants contributing to the temporal maintenance in the non‐Berberis zone. Our study hence demonstrated the contribution of both sexual recombination and off‐season oversummering survival to the temporal maintenance of the pathogen. These new insights into the population biology of PST highlight the general usefulness of the analytical approach proposed.     

Can Norway spruce trees be ‘vaccinated’ against attack by Ips typographus?

1 A field experiment was carried out to test the hypothesis that treatment of Norway spruce trees with the Ips typographus-transmitted blue-stain fungus Ceratocystis polonica enhances tree resistance to later mass attack by this bark beetle. 2 Twenty-five mature trees were pretreated by inoculating a non-lethal dose of the fungus into the bark, while 18 trees served as untreated controls. Three and a half weeks after treatment a bark beetle attack was initiated by attaching dispensers with I. typographus pheromone to the tree trunks. 3 A significantly larger proportion (67%) of the control trees than of the pretreated trees (36%) were killed by the beetle attack. The result is discussed in relation to recent results regarding defence mechanisms in Norway spruce trees.     

Host–parasite genotypic interactions in the honey bee: the dynamics of diversity

Parasites are thought to be a major driving force shaping genetic variation in their host, and are suggested to be a significant reason for the maintenance of sexual reproduction. A leading hypothesis for the occurrence of multiple mating (polyandry) in social insects is that the genetic diversity generated within‐colonies through this behavior promotes disease resistance. This benefit is likely to be particularly significant when colonies are exposed to multiple species and strains of parasites, but host–parasite genotypic interactions in social insects are little known. We investigated this using honey bees, which are naturally polyandrous and consequently produce genetically diverse colonies containing multiple genotypes (patrilines), and which are also known to host multiple strains of various parasite species. We found that host genotypes differed significantly in their resistance to different strains of the obligate fungal parasite that causes chalkbrood disease, while genotypic variation in resistance to the facultative fungal parasite that causes stonebrood disease was less pronounced. Our results show that genetic variation in disease resistance depends in part on the parasite genotype, as well as species, with the latter most likely relating to differences in parasite life history and host–parasite coevolution. Our results suggest that the selection pressure from genetically diverse parasites might be an important driving force in the evolution of polyandry, a mechanism that generates significant genetic diversity in social insects.     

THE JACK OF ALL TRADES IS MASTER OF NONE: A PATHOGEN'S ABILITY TO INFECT A GREATER NUMBER OF HOST GENOTYPES COMES AT A COST OF DELAYED REPRODUCTION

A trade‐off between a pathogen's ability to infect many hosts and its reproductive capacity on each host genotype is predicted to limit the evolution of an expanded host range, yet few empirical results provide evidence for the magnitude of such trade‐offs. Here, we test the hypothesis for a trade‐off between the number of host genotypes that a fungal pathogen can infect (host genotype range) and its reproductive capacity on susceptible plant hosts. We used strains of the oat crown rust fungus that carried widely varying numbers of virulence (avr) alleles known to determine host genotype range. We quantified total spore production and the expression of four pathogen life‐history stages: infection efficiency, time until reproduction, pustule size, and spore production per pustule. In support of the trade‐off hypothesis, we found that virulence level, the number of avr alleles per pathogen strain, was correlated with significant delays in the onset of reproduction and with smaller pustule sizes. Modeling from our results, we conclude that trade‐offs have the capacity to constrain the evolution of host genotype range in local populations. In contrast, long‐term trends in virulence level suggest that the continued deployment of resistant host lines over wide regions of the United States has generated selection for increased host genotype range.     

Orchestration of sexual reproduction and virulence by the fungal mating-type locus

The mating-type locus (MAT) orchestrates sexual reproduction in fungi. Sexual reproduction is related not only to fitness of an organism, but also correlated with virulence in certain pathogens. In the dandruff-associated fungus Malassesia globosa, although the sexual cycle remains to be discovered, whole genome analysis has led to the hypothesis that mating may occur on host skin. Furthermore, the MAT locus of M. globosa and U. hordei provides evidence that transitions between tetrapolar and bipolar systems have independently occurred. These results, together with studies recapitulating the ancestral tetrapolar mating system in Cryptococcus and the structure of MAT in related smut fungi, have furthered understanding on transitions between different mating systems and the evolution of MAT in the Basidiomycota.     

Genetic diversity of the green spruce aphid (Elatobium abietinum Walker) in north-west Europe

Abstract 1 The Random Amplified Polymorphic DNA (RAPD) method was used to investigate genetic diversity of anholocyclic populations of the green spruce aphid, Elatobium abietinum Walker, in north‐west Europe. 2 The results showed that the aphid in this region was divided genetically into three major groups. Aphids from the British Isles and north‐west France comprised the first group, the second group consisted of aphids from Denmark and Iceland, and the third group consisted of aphids from Norway. 3 The results indicated a significant level of gene flow within and between sites and geographical regions, especially in the British Isles and north‐west France. Lateral migration of the aphid and/or sexual reproduction is likely to have facilitated the gene flow. 4 The implications of these findings on management of the green spruce aphid are discussed.     

Estimating the spontaneous mutation rate of loss of sex in the human pathogenic fungus Cryptococcus neoformans

Few events have evolutionary consequences as pervasive as changes in reproductive behavior. Among those changes, the loss of the ability to undergo sexual reproduction is probably the most profound. However, little is known about the rate of loss of sex. Here I describe an experimental system using the fungus Cryptococcus neoformans and provide the first empirical estimate of the spontaneous mutation rate of loss of sex in fungi. Two critical steps in sexual reproduction in C. neoformans were examined: mating and filamentation. Mating, the fusion of cells of opposite sexes, is a universal first step in eukaryotic sexual reproduction. In contrast, filamentation, a prerequisite process preceding meiosis and sexual spore development, is restricted to C. neoformans and a few other fungal species. After approximately 600 mitotic divisions under favorable asexual growth conditions, mean abilities for mating and filamentation decreased significantly by >67 and 24%, respectively. Similarly, though statistically not significant, the mean vegetative growth rates also decreased and among the mutation accumulation lines, the vegetative growth rates were negatively correlated to the mating ability. The estimated mutation rates to decreases in mating ability and filamentation were in excess of 0.0172 and 0.0036, respectively. The results show that C. neoformans can be a highly attractive model for analyses of reproductive system evolution in fungi.