Molecular identification of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila

Traditional phenotypic methods and commercial kits based on carbohydrate assimilation patterns are unable to consistently distinguish among isolates of Pichia guilliermondii, Debaryomyces hansenii and Candida palmioleophila. As result, these species are often misidentified. In this work, we established a reliable method for the identification/differentiation of these species. Our assay was validated by DNA sequencing of the polymorphic region used in a real-time PCR assay driven by species-specific probes targeted to the fungal ITS 1 region. This assay provides a new tool for pathogen identification and for epidemiological, drug resistance and virulence studies of these organisms.     

Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts.

The fish pathogen, Aeromonas salmonicida, could be detected only by bacteriological culture from the kidney of dead or moribund fish in one tank in a hatchery rearing Atlantic salmon (Salmo salar L.) smolts. However, by using a DNA probe specific for this species, allied to a PCR assay, the pathogen could be detected in water, feces and effluent samples taken from this fish tank. Also, the presence of the pathogen was found in effluent samples from two fish tanks containing apparently healthy fish. Subsequently, the presence of pathogen in these tanks was confirmed by an increase in the daily mortality rate and by a plate culture from moribund fish.     

Faster, simpler, more-specific methods for improved molecular detection of Phytophthora ramorum in the field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

Faster, Simpler, More-Specific Methods for Improved Molecular Detection of Phytophthora ramorum in the Field

Phytophthora ramorum is the causal agent of sudden oak death. The pathogen also affects a wide range of tree, shrub, and herbaceous species in natural and landscaped environments as well as plants in the nursery industry. A TaqMan real-time PCR method for the detection of this pathogen in the field has been described previously; this paper describes the development of a number of assays based on this method which have various advantages for use in the field. A scorpion real-time PCR assay that is twice as fast as TaqMan was developed, allowing the detection of P. ramorum in less than 30 min. Also designed was a loop-mediated isothermal amplification (LAMP) assay, which allowed sensitive and specific detection of P. ramorum in 45 min using only a heated block. A positive reaction was identified by the detection of the LAMP product by color change visible to the naked eye.     

长白山阔叶红松林不同采伐强度与森林病害的发生

长白山阔叶红松林不同采伐强度与森林病害的发生袁志文,王庆礼,代力民,钟兆康,赵敏（中国科学院沈阳应用生态研究所１１００１５）ＤｉｆｆｅｒｅｎｔＣｕｔｔｉｎｇｉｎｔｅｎｓｉｔｉｅｓａｎｄＩｎｃｉｄｅｎｃｅｏｆＦｏｒｅｓｔＤｉｓｃａｓｅｓｉｎＢｒｏａｄＬ...     

Polymerase Chain Reaction Assay for Rapid, Sensitive Detection, and Identification of Colletotrichum gloeosporioides causing Greater Yam Anthracnose

Anthracnose caused by Colletotrichum gloeosporioides is an economically important disease which affects greater yam (Dioscorea alata L.) worldwide. Apart from airborne conidia, the pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of C. gloeosporioides in soil and planting material. In conventional (single-round) PCR, the limit of detection was 20?pg, whereas in nested PCR the detection limit increased to 0.2?pg of DNA. The primers designed were found to be highly specific and could be used for accurate identification of the pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.     

Identification of two GH18 chitinase family genes and their use as targets for detection of the crayfish-plague oomycete Aphanomyces astaci

Background  

The oomycete Aphanomyces astaci is regarded as the causative agent of crayfish plague and represents an evident hazard for European crayfish species. Native crayfish populations infected with this pathogen suffer up to 100% mortality. The existence of multiple transmission paths necessitates the development of a reliable, robust and efficient test to detect the pathogen. Currently, A. astaci is diagnosed by a PCR-based assay that suffers from cross-reactivity to other species. We developed an alternative closed-tube assay for A. astaci, which achieves robustness through simultaneous amplification of multiple functionally constrained genes.     

Species‐Specific PCR‐Based Assay for Identification and Detection of Phomopsis (Diaporthe) azadirachtae Causing Die‐Back Disease in Azadirachta indica

Die‐back disease caused by Phomopsis (Diaporthe) azadirachtae is the devastating disease of Azadirachta indica. Accurate identification of P. azadirachtae is always problematic due to morphological plasticity and delayed appearance of conidia. A species‐specific PCR‐based assay was developed for rapid and reliable identification of P. azadirachtae by designing a species‐specific primer‐targeting ITS region of P. azadirachtae isolates. The assay was validated with DNA isolated from different Phomopsis species and other fungal isolates. The PCR assay amplified 313‐bp product from all the isolates of P. azadirachtae and not from any other Phomopsis species or any genera indicating its specificity. The assay successfully detected the pathogen DNA in naturally and artificially infected neem seeds and twigs indicating its applicability in seed quarantine and seed health testing. The sensitivity of the assay was 100 fg when genomic DNA of all isolates was analysed. The PCR‐based assay was 92% effective in comparison with seed plating technique in detecting the pathogen. This is the first report on the development of species‐specific PCR assay for identification and detection of P. azadirachtae. Thus, PCR‐based assay developed is very specific, rapid, confirmatory and sensitive tool for detection of pathogen P. azadirachtae at early stages.     

Morphological and molecular characterization of a Hirsutella species infecting the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), in Florida

The Asian citrus psyllid, Diaphorina citri Kuwayama, is an invasive pest that vectors citrus greening disease, which recently was detected in Florida. Mycosed adult D. citri were collected at four sites in central Florida between September 2005 and February 2006. Observation of the cadavers using scanning electron microscopy revealed that the pathogen had branched synnemata supporting monophiladic conidiogenous cells. A high-fidelity polymerase chain reaction (PCR) assay was used to amplify the 18S rRNA, 28S rRNA and beta-tubulin genes of the pathogen for phylogenetic analysis. The morphological and genetic data indicated that the pathogen was a novel isolate related to Hirsutella citriformis Speare. PCR assays using isolate-specific primers designed from the unique putative intron region of the beta-tubulin sequence distinguished the psyllid pathogen from five related Hirsutella species. The pathogen was maintained in vivo by exposing healthy D. citri to the synnemata borne on field-collected cadavers. Infected psyllids had an abundance of septate hyphal bodies in their hemolymph and exhibited behavioral symptoms of disease. In vitro cultures of the pathogen were slow-growing and produced synnemata similar to those found on mycosed D. citri. In laboratory bioassays, high levels of mortality were observed in D. citri that were exposed to the conidia-bearing synnemata produced in vivo and in vitro.     

Dioecy,hermaphrodites and pathogen load in plants

Sex‐specific investment in pathogen resistance and immunity has been widely reported in animals and to a much lesser degree in plants. Here, we investigated the incidence of fungal pathogens in dioecious versus hermaphroditic plant species. We found that direct studies on differences between males and females in disease resistance or pathogen incidence were rare or non‐existent in plants, but if we made the prediction that if such differences exist (e.g. if males are less resistant than females), dioecious species should have a higher variation in pathogen diversity than hermaphrodites. Comparative studies on paired dioecious and hermaphrodite species from multiple plant families showed that hermaphrodites had a higher average pathogen load than dioecious species, consistent with the idea that higher outcrossing is beneficial to resistance to a greater diversity of pathogens. There was however no support for dioecious species also having a greater variance in pathogen diversity. Our results are consistent with dioecy providing a benefit in terms of pathogen resistance, but the data were insufficient to resolve if the male and female plants showed sex‐specific investment in resistance.     

Effects of elevated CO2, nitrogen deposition, and decreased species diversity on foliar fungal plant disease

Three components of global change, elevated CO₂, nitrogen addition, and decreased plant species richness (‘diversity’), increased the percent leaf area infected by fungi (pathogen load) for much to all of the plant community in one year of a factorial grassland experiment. Decreased plant diversity had the broadest effect, increasing pathogen load across the plant community. Decreased diversity increased pathogen load primarily by allowing remaining plant species to increase in abundance, facilitating spread of foliar fungal pathogens specific to each plant species. Changes in plant species composition also strongly influenced community pathogen load, with communities that lost less disease prone plant species increasing more in pathogen load. Elevated CO₂ increased pathogen load of C₃ grasses, perhaps by decreasing water stress, increasing leaf longevity, and increasing photosynthetic rate, all of which can promote foliar fungal disease. Decreased plant diversity further magnified the increase in C₃ grass pathogen load under elevated CO₂. Nitrogen addition increased pathogen load of C₄ grasses by increasing foliar nitrogen concentration, which can enhance pathogen infection, growth, and reproduction. Because changes in foliar fungal pathogen load can strongly influence grassland ecosystem processes, our study suggests that increased pathogen load can be an important mechanism by which global change affects grassland ecosystems.     

Cryptic species, native populations and biological invasions by a eucalypt forest pathogen

Human‐associated introduction of pathogens and consequent invasions is very evident in areas where no related organisms existed before. In areas where related but distinct populations or closely related cryptic species already exist, the invasion process is much harder to unravel. In this study, the population structure of the Eucalyptus leaf pathogen Teratosphaeria nubilosa was studied within its native range in Australia, including both commercial plantations and native forests. A collection of 521 isolates from across its distribution was characterized using eight microsatellite loci, resulting in 112 multilocus haplotypes (MLHs). Multivariate and Bayesian analyses of the population conducted in structure revealed three genetically isolated groups (A, B and C), with no evidence for recombination or hybridization among groups, even when they co‐occur in the same plantation. DNA sequence data of the ITS (n = 32), β‐tubulin (n = 32) and 27 anonymous loci (n = 16) were consistent with microsatellite data in suggesting that T. nubilosa should be considered as a species complex. Patterns of genetic diversity provided evidence of biological invasions by the pathogen within Australia in the states of Western Australia and New South Wales and helped unravel the pattern of invasion beyond Australia into New Zealand, Brazil and Uruguay. No significant genetic differences in pathogen populations collected in native forests and commercial plantations were observed. This emphasizes the importance of sanitation in the acquisition of nursery stock for the establishment of commercial plantations.     

Rapid and Sensitive Detection of Sclerotium rolfsii Associated with Collar Rot Disease of Amorphophallus paeoniifolius by Species-Specific Polymerase Chain Reaction Assay

Collar rot disease caused by Sclerotium rolfsii is an economically important disease prevailing in all Amorphophallus growing areas. The pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of S. rolfsii in soil and planting material. The PCR detection limit was 10 pg in conventional assay whereas 0.1 pg in nested assay. The primers designed were found to be highly specific and could be used for accurate identification of pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.     

Domestication of maize, sorghum, and sugarcane did not drive the divergence of their smut pathogens

We investigated two alternative hypotheses for the origin of crop pathogen species: that human-mediated agricultural practices drove the divergence of many crop plant pathogen species or that coevolutionary processes in natural populations of the crops' ancestors drove divergence of pathogen species. We distinguished between these two hypotheses by constructing a robust multigene phylogeny and estimating the dates of divergence among four, monophyletic species of smut fungi (Ustilago maydis, U. scitaminea, Sporisorium reilianum, S. sorghi) known to specifically infect maize, sorghum, sugarcane, and their wild ancestors. Without a fossil record for smut fungi, we calibrated the pathogen species' divergence times to their plant host divergence times. Specifically, a calibration date of 10,000 years was employed to test the hypothesis that the fungal species originated at the time of domestication of their current hosts and a calibration date of 50 million years was employed to test the hypothesis that the fungal species originated on wild ancestors of their domesticated hosts. Substitution rates at five protein coding genes were calculated and rates obtained for the 10,000 year calibration date were orders of magnitude faster than those commonly reported for eukaryotes, thus rejecting the hypothesis that these smut pathogen species diverged at the time of domestication. In contrast, substitution rates obtained for the 50 million year calibration were comparable to eukaryotic substitution rates. We used the 50 million year calibration to estimate divergence times of taxa in two datasets, one comprised solely the focal species and one comprised the focal species and additional related taxa. Both datasets indicate that all taxa diverged millions of years ago, strongly supporting the hypothesis that smut species diverged before the time of domestication and modern agriculture. Thus, smut species diverged in the ecological context of natural host plant and fungal populations.     

Pathogen interactions, population cycles, and phase shifts

Interspecific pathogen interactions can profoundly affect pathogen population dynamics and the efficacy of control strategies. However, many pathogens exhibit cyclic abundance patterns (e.g., seasonality), and temporal asynchrony between interacting pathogens could reduce the impact of those interactions. Here we use an extension of our previously published model to investigate the effects of cycles on pathogen interaction. We demonstrate that host immune memory can maintain the impact of an interaction, even when the effector pathogen abundance is low or the pathogen is absent. Paradoxically, immune memory can result in pathogens interacting more strongly when temporally out of phase. We find that interactions between species can result in changes to the temporal pattern of the affected species. We further demonstrate that this may be observed in a natural host-pathogen system. Given the continuing debate regarding the relevance of pathogen interactions in natural systems and increasing concern about treatment strategies for coinfections, both the discovery of a shift in cycle in empirical data and the mechanism by which we identified it are important. Finally, because the model structure used here is analogous to models of a simple predator-prey system, we also consider the consequences of these findings in the context of that system.     

A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata,Babesia bovis and Anaplasma marginale in cattle

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.