Virulence Phenotyping and Molecular Genotyping Reveal High Diversity Within and Strong Gene Flow Between the Puccinia striiformis f. sp. tritici Populations Collected from Barberry and Wheat in Shaanxi Province of China.

Emergence of new Puccinia striiformis f. sp. tritici races that overcome resistance of wheat cultivars is a challenging issue for wheat production. Although sexual reproduction of the fungus on barberry plants under field conditions in the spring in China has been reported, the diversity of the pathogen on barberry plants and the relationship to the population in wheat fields have not been determined. In the present study, two P. striiformis f. sp. tritici populations collected in western Shaanxi Province in May 2016, one from barberry plants (103 isolates) and the other from nearby wheat crops (107 isolates), were phenotyped for virulence and genotyped with simple sequence repeat (SSR) markers. The phenotypic and genotypic data of the two populations were compared to determine their relationships. A total of 120 races, including 29 previously known races (seven were shared by the two populations) and 91 new races (35 from barberry and 56 from wheat), were identified. Similarly, a total of 132 multilocus genotypes, including 51 only from barberry, 77 only from wheat, and four from both, were detected using the SSR markers. Analyses of molecular variance identified high (93%) genetic variance within populations and low but still significant variance (7%) between the populations. Nonparametric multivariate discriminant analysis of principal components and STRUCTURE analysis showed that the two populations had a close relationship with little genetic differentiation (FST = 0.038) and strong gene flow (Nm = 6.34, P = 0.001) between them. Although the analysis of linkage disequilibrium indicated clonal populations, the isolation of P. striiformis f. sp. tritici from barberry plants and the high genetic diversities in the barberry and wheat populations suggest that barberry plants provide aeciospores to infect wheat crops in the area. The information is useful for understanding stripe rust epidemiology and management of the disease.

Direct Evidence Demonstrates that Puccinia striiformis f. sp. tritici Infects Susceptible Barberry to Complete Sexual Cycle in Autumn.

Wheat stripe rust is an airborne and destructive disease caused by a heteroecious rust fungus Puccinia striiformis f. sp. tritici (Pst). Studies have demonstrated that the rust pathogen accomplishes sexual reproduction on susceptible barberry under natural conditions in spring, whereas Pst infection on barberry is still in blank in other seasons. In late October 2016, aecial production on barberry shrubs were observed in Linzhi, Tibet, China. Therefore, experimental tests were conducted to verify the existence of sexual cycles of Pst in this season. By inoculating 52 aecial clusters from 30 rusted barberry leaves, four Pst samples, T1 to T4, were successfully recovered from the rusted barberry shrubs. Sixty-five single uredinium (SU) isolates were derived from the four Pst samples. Based on virulence tests on the Chinese differential hosts, T1 to T4 samples were unknown races and showed mixed reactions on some differentials. Twenty-one known races and 44 unknown races belonging to five race groups were identified among the 65 SU isolates. Meanwhile, the 65 SU isolates produced 26 various virulence patterns (VPs; called VP1-VP26) on 25 single Yr gene lines and 15 multilocus genotypes (MLGs) at nine simple sequence repeat marker loci. Clustering analysis showed similar lineage among subpopulations and different lineage between subpopulations. Linkage disequilibrium analysis indicated that the SU population was produced sexually. This study first reported that Pst infects susceptible barberry to complete sexual reproduction in autumn. The results update the knowledge of disease cycle and management of wheat stripe rust and contribute to the understanding of rust genetic diversity in Tibet.

Identification of Mahonia Species as Alternate Hosts for Puccinia striiformis f. sp. tritici and Determination of Existence of Sexual Propagation of the Rust Pathogen on Mahonia Under Natural Conditions in China.

Many Berberis species have been identified as alternate hosts for Puccinia striiformis f. sp. tritici. Importantly, susceptible Berberis species are determined to play an important role in the occurrence of sexual reproduction, generation of new races of the rust pathogen. However, little is known about Mahonia serving as alternate hosts for P. striiformis f. sp. tritici and their role to commence sexual reproduction of the rust fungus under natural conditions. Herein, three Mahonia species or subspecies, Mahonia fortunei, M. eurybracteata subsp. ganpinensis, and M. sheridaniana, were identified as alternate hosts for P. striiformis f. sp. tritici, and seven Mahonia species were highly resistant to the rust pathogen. We recovered seven samples of P. striiformis f. sp. tritici from naturally rusted Mahonia cardiophylla plants. Totally, 54 single uredinium (SU) isolates, derived from the seven samples, generated 20 different race types, including one known race type, and 19 new race types. SNP markers analysis showed that all SU isolates displayed high phenotype diversity (H = 0.32) with a high Shannon's information index (I = 0.49). Analysis of linkage disequilibrium indicated an insignificant rbarD value (rbarD = 0.003, P < 0.1). As a result, all SU isolates are sexually produced, suggesting that P. striiformis f. sp. tritici parasitizes susceptible Mahonia to complete sexual reproduction under natural conditions. The role of Mahonia in occurrence of wheat stripe rust are needed to study for management of the disease.

Distinct Transcriptomic Reprogramming in the Wheat Stripe Rust Fungus During the Initial Infection of Wheat and Barberry.

Puccinia striiformis f. sp. tritici is the causal agent of wheat stripe rust that causes severe yield losses all over the world. As a macrocyclic heteroecious rust fungus, it is able to infect two unrelated host plants, wheat and barberry. Its urediniospores infect wheat and cause disease epidemic, while its basidiospores parasitize barberry to fulfill the sexual reproduction. This complex life cycle poses interesting questions on the different mechanisms of pathogenesis underlying the infection of the two different hosts. In the present study, transcriptomes of P. striiformis f. sp. tritici during the initial infection of wheat and barberry leaves were qualitatively and quantitatively compared. As a result, 142 wheat-specifically expressed genes (WEGs) were identified, which was far less than the 2,677 barberry-specifically expressed genes (BEGs). A larger proportion of evolutionarily conserved genes were observed in BEGs than that in WEGs, implying a longer history of the interaction between P. striiformis f. sp. tritici and barberry. Additionally, P. striiformis f. sp. tritici differentially expressed genes (DEGs) between wheat at 1 and 2 days postinoculation (dpi) and barberry at 3 and 4 dpi were identified by quantitative analysis. Gene Ontology analysis of these DEGs and expression patterns of P. striiformis f. sp. tritici pathogenic genes, including those encoding candidate secreted effectors, cell wall-degrading enzymes, and nutrient transporters, demonstrated that urediniospores and basidiospores exploited distinct strategies to overcome host defense systems. These results represent the first analysis of the P. striiformis f. sp. tritici transcriptome in barberry and contribute to a better understanding of the evolutionary processes and strategies of different types of rust spores during the infection process on different hosts.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

An Avirulence Gene Cluster in the Wheat Stripe Rust Pathogen (Puccinia striiformis f. sp. tritici) Identified through Genetic Mapping and Whole-Genome Sequencing of a Sexual Population.

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2 Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (<200 kb). The discovery of the Avr gene cluster is useful for understanding pathogen evolution, and the identification of candidate genes is an important step toward cloning Avr genes for studying molecular mechanisms of pathogen-host interactions.IMPORTANCE Stripe rust is a destructive disease of wheat worldwide. Growing resistant cultivars is the most effective, easy-to-use, economical, and environmentally friendly strategy for the control of the disease. However, P. striiformis f. sp. tritici can produce new virulent races that may circumvent race-specific resistance. Therefore, understanding the genetic basis of the interactions between wheat genes for resistance and P. striiformis f. sp. tritici genes for avirulence is useful for improving cultivar resistance for more effective control of the disease. This study developed a high-quality map that facilitates genomic and genetic studies of important traits related to pathogen pathogenicity and adaptation to different environments and crop cultivars carrying different resistance genes. The information on avirulence/virulence genes identified in this study can be used for guiding breeding programs to select combinations of genes for developing new cultivars with effective resistance to mitigate this devastating disease.

Mapping non-host resistance to the stem rust pathogen in an interspecific barberry hybrid.

BACKGROUND: Non-host resistance (NHR) presents a compelling long-term plant protection strategy for global food security, yet the genetic basis of NHR remains poorly understood. For many diseases, including stem rust of wheat [causal organism Puccinia graminis (Pg)], NHR is largely unexplored due to the inherent challenge of developing a genetically tractable system within which the resistance segregates. The present study turns to the pathogen's alternate host, barberry (Berberis spp.), to overcome this challenge. RESULTS: In this study, an interspecific mapping population derived from a cross between Pg-resistant Berberis thunbergii (Bt) and Pg-susceptible B. vulgaris was developed to investigate the Pg-NHR exhibited by Bt. To facilitate QTL analysis and subsequent trait dissection, the first genetic linkage maps for the two parental species were constructed and a chromosome-scale reference genome for Bt was assembled (PacBio + Hi-C). QTL analysis resulted in the identification of a single 13 cM region (~ 5.1 Mbp spanning 13 physical contigs) on the short arm of Bt chromosome 3. Differential gene expression analysis, combined with sequence variation analysis between the two parental species, led to the prioritization of several candidate genes within the QTL region, some of which belong to gene families previously implicated in disease resistance. CONCLUSIONS: Foundational genetic and genomic resources developed for Berberis spp. enabled the identification and annotation of a QTL associated with Pg-NHR. Although subsequent validation and fine mapping studies are needed, this study demonstrates the feasibility of and lays the groundwork for dissecting Pg-NHR in the alternate host of one of agriculture's most devastating pathogens.

Tackling the re-emergence of wheat stem rust in Western Europe.

In our recent Communications Biology article, we reported the first occurrence of wheat stem rust in the UK in nearly six decades. An increased incidence of wheat stem rust in Western Europe, caused by the fungus Puccinia graminis f. sp. tritici, could signify the return of this formidable foe. As pathologists fight back against this devastating disease we outline the continuing research and strategies being employed to bridle its onslaught.

Identification of Berberis Species Collected from the Himalayan Region of Pakistan Susceptible to Puccinia striiformis f. sp. tritici.

Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen infecting cereal crops and grasses, was believed to have a hemicyclic life cycle consisting of uredinial and telial stages before the recent discovery of barberry (Berberis spp.) as an alternate (aecial) host for the fungus. This discovery has improved the understanding of the biology of the stripe rust pathogen. The Himalayan and near-Himalayan regions of Pakistan, China, and Nepal are considered as the center of diversity for Pst pathogen. High genetic diversity has been reported in these areas, probably resulting from the sexual reproduction of the stripe rust fungus. To determine if Berberis species growing in Pakistan are susceptible to Pst, we collected seeds of five species and two subspecies from the Himalayan region in 2016 and inoculated the seedlings with germinated teliospores of a Pakistani Pst isolate under controlled conditions. Pycnia and aecia were produced on all inoculated plants of these species and subspecies, and were demonstrated as Pst by successful infection of wheat plants with aeciospores. This study showed that the tested Pakistani Berberis species and subspecies are susceptible to Pst under controlled conditions.

The Barberry Eradication Program in Minnesota for Stem Rust Control: A Case Study.

The Barberry Eradication Program was an unprecedented federal and state cooperative plant disease control campaign between 1918 and the late 1970s to remove common barberry ( Berberis vulgaris), the alternate host of Puccinia graminis f. sp. tritici, from the major centers of wheat production in the United States. Eradication of barberry has been credited with helping to reduce stem rust of wheat to a minor problem in the United States by the end of the campaign. The Barberry Eradication Program has also been viewed as a model for successful eradication based on its robust leadership, effective publicity and public cooperation, forceful quarantine laws, and adaptive eradication methods and procedures employed in its field operations. The Barberry Eradication Program was particularly successful because of its leaders' ability to adapt to changing internal and external conditions over time. The program lasted nearly a century, extending through two world wars and the Great Depression, with each period producing unique challenges. Because of its central role, barberry eradication in Minnesota offers an excellent case study to examine how the program developed over time and ultimately achieved success.

An interspecific barberry hybrid enables genetic dissection of non-host resistance to the stem rust pathogen Puccinia graminis.

Stem rust, caused by Puccinia graminis (Pg), remains a devastating disease of wheat, and the emergence of new Pg races virulent on deployed resistance genes fuels the ongoing search for sources of durable resistance. Despite its intrinsic durability, non-host resistance (NHR) is largely unexplored as a protection strategy against Pg, partly due to the inherent challenge of developing a genetically tractable system within which NHR segregates. Here, we demonstrate that Pg's far less studied ancestral host, barberry (Berberis spp.), provides such a unique pathosystem. Characterization of a natural population of B. ×ottawensis, an interspecific hybrid of Pg-susceptible B. vulgaris and Pg-resistant B. thunbergii (Bt), reveals that this uncommon nothospecies can be used to dissect the genetic mechanism(s) of Pg-NHR exhibited by Bt. Artificial inoculation of a natural population of B. ×ottawensis accessions, verified via genotyping by sequencing to be first-generation hybrids, revealed 51% susceptible, 33% resistant, and 16% intermediate phenotypes. Characterization of a B. ×ottawensis full sib family excluded the possibility of maternal inheritance of the resistance. By demonstrating segregation of Pg-NHR in a hybrid population, this study challenges the assumed irrelevance of Bt to Pg epidemiology and lays a novel foundation for the genetic dissection of NHR to one of agriculture's most studied pathogens.

Inheritance of Virulence, Construction of a Linkage Map, and Mapping Dominant Virulence Genes in Puccinia striiformis f. sp. tritici Through Characterization of a Sexual Population with Genotyping-by-Sequencing.

Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen, is a dikaryotic, biotrophic, and macrocyclic fungus. Genetic study of P. striiformis f. sp. tritici virulence was not possible until the recent discovery of Berberis spp. and Mahonia spp. as alternate hosts. To determine inheritance of virulence and map virulence genes, a segregating population of 119 isolates was developed by self-fertilizing P. striiformis f. sp. tritici isolate 08-220 (race PSTv-11) on barberry leaves under controlled greenhouse conditions. The progeny isolates were phenotyped on a set of 29 wheat lines with single genes for race-specific resistance and genotyped with simple sequence repeat (SSR) markers, single nucleotide polymorphism (SNP) markers derived from secreted protein genes, and SNP markers from genotyping-by-sequencing (GBS). Using the GBS technique, 10,163 polymorphic GBS-SNP markers were identified. Clustering and principal component analysis grouped these markers into six genetic groups, and a genetic map, consisting of six linkage groups, was constructed with 805 markers. The six clusters or linkage groups resulting from these analyses indicated a haploid chromosome number of six in P. striiformis f. sp. tritici. Through virulence testing of the progeny isolates, the parental isolate was found to be homozygous for the avirulence loci corresponding to resistance genes Yr5, Yr10, Yr15, Yr24, Yr32, YrSP, YrTr1, Yr45, and Yr53 and homozygous for the virulence locus corresponding to resistance gene Yr41. Segregation was observed for virulence phenotypes in response to the remaining 19 single-gene lines. A single dominant gene or two dominant genes with different nonallelic gene interactions were identified for each of the segregating virulence phenotypes. Of 27 dominant virulence genes identified, 17 were mapped to two chromosomes. Markers tightly linked to some of the virulence loci may facilitate further studies to clone these genes. The virulence genes and their inheritance information are useful for understanding the host-pathogen interactions and for selecting effective resistance genes or gene combinations for developing stripe rust resistant wheat cultivars.

Genetic diversity within and among aecia of the wheat rust fungus Puccinia striiformis on the alternate host Berberis vulgaris.

An isolate of the fungus Puccinia striiformis, causing yellow (stripe) rust on cereals and grasses, was selfed on the alternate (sexual) host, Berberis vulgaris. This enabled us to investigate genetic variability of progeny isolates within and among aecia. Nine aecial clusters each consisting of an aecium (single aecial cup) and nine clusters containing multiple aecial cups were selected from 18 B. vulgaris leaves. Aeciospores from each cluster were inoculated on susceptible wheat seedlings and 64 progeny isolates were recovered. Molecular genotyping using 37 simple sequence repeat markers confirmed the parental origin of all progeny isolates. Thirteen molecular markers, which were heterozygous in the parental isolate, were used to analyse genetic diversity within and among aecial cups. The 64 progeny isolates resulted in 22 unique recombinant multilocus genotypes and none of them were resampled in different aecial clusters. Isolates derived from a single cup were always of the same genotype whereas isolates originating from clusters containing up to nine aecial cups revealed one to three genotypes per cluster. These results implied that each aecium was the result of a successful fertilization in a corresponding pycnium and that an aecium consisted of genetically identical aeciospores probably multiplied via repetitive mitotic divisions. Furthermore, the results suggested that aecia within a cluster were the result of independent fertilization events often involving genetically different pycniospores. The application of molecular markers represented a major advance in comparison to previous studies depending on phenotypic responses on host plants. The study allowed significant conclusions about fundamental aspects of the biology and genetics of an important cereal rust fungus.

Basidiospores of Puccinia striiformis f. sp. tritici succeed to infect barberry, while Urediniospores are blocked by non-host resistance.

Stripe rust (Yellow rust) caused by Puccinia striiformis f. sp. tritici (Pst) is a major disease of wheat worldwide. The use of resistant cultivars to control Pst has been very effective, low-cost, and ecologically sound. However, virulence patterns of Pst can quickly change, which may render resistant cultivars susceptible. The discovery of infection of Berberis spp. by basidiospores of Pst in 2010 raised important concerns about the evolution of new virulent races of the pathogen. Little is known about the infection process of Berberis spp. by basidiospores of Pst and the interaction between Berberis spp. and asexual urediniospores. In this study, the interaction between Pst urediniospores and Berberis spp. was investigated at histological and cytological levels. Our results indicate that Berberis spp. expresses a continuum of layered defenses comprised of structural and chemical changes in the cell wall as well as post-haustorial hypersensitive responses to urediniospore infection. Our study also re-examines in detail the infection process of Pst basidiospores on Berberis spp. and provides useful information for further research on the molecular mechanisms governing the interaction between Berberis spp. and Pst.

Virulence and Simple Sequence Repeat Marker Segregation in a Puccinia striiformis f. sp. tritici Population Produced by Selfing a Chinese Isolate on Berberis shensiana.

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust, frequently produces new races overcoming resistance in wheat cultivars. A recently identified race, V26 with virulence to Yr26 and many other stripe rust resistance genes, has a high potential to cause epidemics in China. In this study, teliospores from a single-urediniospore isolate of V26 (Pinglan 17-7) produced on the wheat line 92R137 (Yr26) were used to produce a sexual population through selfing by infecting Berberis shensiana plants under controlled conditions. One hundred and eighteen progeny isolates and the parental isolate were phenotyped for virulence/avirulence on 24 Yr gene lines of wheat. These progeny isolates were all avirulent to Yr5, Yr8, Yr15, and YrTr1 and virulent to Yr1, Yr2, Yr7, Yr9, Yr10, Yr17, Yr24, Yr25, Yr26, YrA, YrExp2, and YrV23, indicating that the parental isolate is homozygous avirulent or homozygous virulent at these loci. The progeny population segregated for avirulence to Yr6, Yr43, and YrSP at one locus (3 avirulent:1 virulent ratio); for virulence to Yr27 and Yr28 at one locus (3 virulent:1 avirulent); and for Yr4, Yr32, and Yr44 at two loci (15 virulent:1 avirulent). Among the eight segregating avirulence/virulence loci, association was found between virulence to Yr4 and Yr32, as well as between virulence to Yr6 and Yr43 based on χ(2) tests. From 82 genotypically different progeny isolates, 24 pathotypes and 82 multilocus genotypes were identified. The results show that a highly diverse population can be produced from a single isolate by selfing on a barberry plant and sexually produced population can be used to genetically characterize virulence of the stripe rust pathogen.

Sexual structures and recombination of the wheat rust fungus Puccinia striiformis on Berberis vulgaris.

An isolate of the basidiomycete Puccinia striiformis, which causes yellow (stripe) rust on wheat, was selfed on the newly discovered alternate host, Berberis vulgaris. This allowed a study of the segregation of molecular markers and virulence in the progeny isolates, and of the development of fungal sexual structures and spore forms. Pycnia and aecia were obtained after inoculation of B. vulgaris with basidiospores resulting from germinating teliospores from infected wheat leaves. Subsequent inoculation of wheat with aeciospores from bulked aecia resulted in 16 progeny isolates of the S1 generation. Genotyping with 42 simple sequence repeat (SSR) markers confirmed a parental origin of progeny isolates. Of the 42 analyzed loci, 15 were heterozygous in the parental isolate and 14 revealed segregation in the progenies. This resulted in 11 new multilocus genotypes (MLGs), which confirmed segregation following sexual reproduction. Additionally, parental and progeny isolates were phenotyped using a genetic stock of wheat genotypes representing 21 resistance genes. All S1 progeny isolates had virulence for 14 out of 15 loci where the parental isolate was virulent. This was consistent with the hypothesis that virulence in plant pathogens is often recessive to avirulence, i.e., only expressed in a homozygous state. Furthermore, no segregation was observed for five out of six loci, for which the parental isolate had an avirulent phenotype. The results for one of the two segregating virulence/avirulence loci suggested that the parental isolate was heterozygous with Avr alleles resulting in different but clearly avirulent phenotypes. The other locus indicated that additional genes modifying the phenotypic expression of avirulence were involved.

Virulence and Molecular Analyses Support Asexual Reproduction of Puccinia striiformis f. sp. tritici in the U.S. Pacific Northwest.

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, occurs every year and causes significant yield losses in the U.S. Pacific Northwest (PNW). A large number of P. striiformis f. tritici races are identified every year and predominant races have changed rapidly. Barberry and mahonia plants, which have been identified under controlled conditions as alternate hosts for the fungus, are found in the region. However, whether sexual reproduction occurs in the P. striiformis f. sp. tritici population under natural conditions is not clear. To determine the reproduction mode of the P. striiformis f. sp. tritici population using virulence and molecular markers, a systematic collection of leaf samples with a single stripe of uredia was made in 26 fields in the PNW in 2010. In total, 270 isolates obtained from the PNW collection, together with 66 isolates from 20 other states collected in the same year, were characterized by virulence tests and simple sequence repeat (SSR) markers. In total, 21 races and 66 multilocus genotypes (MLGs) were detected, of which 15 races and 32 MLGs were found in the PNW. Cluster analysis with the SSR marker data revealed two genetic groups, which were significantly correlated to the two virulence groups. The analyses of genotype/individual ratio, multilocus linkage disequilibrium, and heterozygosity strongly supported asexual reproduction for the pathogen population in the PNW, as well as other regions of the United States.

Amplicon pyrosequencing reveals the soil microbial diversity associated with invasive Japanese barberry (Berberis thunbergii DC.).

The soil microbial community acts as a reservoir of microbes that directly influences the structure and composition of the aboveground plant community, promotes plant growth, increases stress tolerance and mediates local patterns of nutrient cycling. Direct interactions between plants and rhizosphere-dwelling microorganisms occur at, or near, the surface of the root. Upon introduction and establishment, invasive plants modify the soil microbial communities and soil biochemistry affecting bioremediation efforts and future plant communities. Here, we used tag-encoded FLX amplicon 454 pyrosequencing (TEFAP) to characterize the bacterial and fungal community diversity in the rhizosphere of Berberis thunbergii DC. (Japanese barberry) from invasive stands in coastal Maine to investigate effects of soil type, soil chemistry and surrounding plant cover on the soil microbial community structure. Acidobacteria, Actinobacteria, Proteobacteria and Verrucomicrobia were the dominant bacterial phyla, whereas fungal communities were comprised mostly of Ascomycota and Basidiomycota phyla members, including Agaricomycetes and Sordariomycetes. Bulk soil chemistry had more effect on the bacterial community structure than the fungal community. An effect of geographic location was apparent in the rhizosphere microbial communities, yet it was less significant than the effect of surrounding plant cover. These data demonstrate a high degree of spatial variation in the rhizosphere microbial communities of Japanese barberry with apparent effects of soil chemistry, location and canopy cover on the microbial community structure.

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.

New species of the genus Metschnikowia isolated from flowers in Indonesia, Metschnikowia cibodasensis sp. nov.

A novel species, Metschnikowia cibodasensis, is proposed to accommodate eight strains (ID03- 0093(T), ID03-0094, ID03-0095, ID03-0096, ID03-0097, ID03-0098, ID03-0099, and ID03-0109) isolated from flowers of Saurauia pendula, Berberis nepalensis, and Brunfelsia americana in Cibodas Botanical Garden, West Java, Indonesia. The type strain of M. cibodasensis is ID03- 0093(T) (= NBRC 101693(T) =UICC Y-335(T) = BTCC-Y25(T)). The common features of M. cibodasensis are a spherical to ellipsoidopedunculate shaped ascus, which contains one or two needleshaped ascospores, and lyse at maturity. Asci generally develop directly from vegetative cells but sometimes from chlamydospores. The neighbor-joining tree based on the D1/D2 domain of nuclear large subunit (nLSU) ribosomal DNA sequences strongly supports that M. cibodasensis (eight strains) and its closest teleomorphic species, M. reukaufii, are different species by a 100% bootstrap value. The type strain of M. cibodasensis, ID03-0093(T), differed from M. reukaufii NBRC 1679(T) by six nt (five substitutions and one deletion) in their D1/D2 region of nLSU rDNA, and by 18 nt (five deletions, four insertions, and nine substitutions) in their internal transcribed spacer regions of rDNA, respectively. Four strains representative of M. cibodasensis (ID03-0093(T), ID03-0095, ID03-0096, and ID03-0099) showed a mol% G+C content of 44.05 ± 0.25%, whereas that of M. reukaufii NBRC 1679(T) was 41.3%. The low value of DNADNA homology (5-16%) in four strains of M. cibodasensis and M. reukaufii NBRC 1679(T) strongly supported that these strains represent a distinct species.

Identification of eighteen Berberis species as alternate hosts of Puccinia striiformis f. sp. tritici and virulence variation in the pathogen isolates from natural infection of barberry plants in China.

ABSTRACT The wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici) population in China has been reported to be a distinct genetic group with higher diversity than those in many other countries. Genetic recombination in the P. striiformis f. sp. tritici population has been identified with molecular markers but whether sexual reproduction occurs in China is unknown. In this study, we surveyed barberry plants for infection by rust fungi in the stripe rust "hotspot" regions in Gansu, Sichuan, and Shaanxi provinces; collected barberry plants and inoculated plants of 20 Berberis spp. with germinated teliospores under controlled greenhouse conditions for susceptibility to P. striiformis f. sp. tritici; and tested P. striiformis f. sp. tritici isolates obtained from aecia on naturally infected barberry plants on the wheat genotypes used to differentiate Chinese P. striiformis f. sp. tritici races to determine virulence variations. Different Berberis spp. were widely distributed and most surveyed plants had pycnia and aecia of rust fungi throughout the surveyed regions. In total, 28 Berberis spp. were identified during our study. From 20 Berberis spp. tested with teliospores of P. striiformis f. sp. tritici from wheat plants, 18 species were susceptible under greenhouse conditions. Among 3,703 aecia sampled from barberry plants of three species (Berberis shensiana, B. brachypoda, and B. soulieana) under natural infections in Gansu and Shaanxi provinces, four produced P. striiformis f. sp. tritici uredinia on susceptible wheat 'Mingxian 169'. Sequence of the internal transcribed spacer (ITS) regions of the four isolates from barberry shared 99% identity with the P. striiformis f. sp. tritici sequences in the National Center for Biotechnology Information database. The four isolates had virulence patterns different from all previously reported races collected from wheat plants. Furthermore, 82 single-uredinium isolates obtained from the four barberry isolates had high virulence diversity rates of 9.0 to 28.1%, indicating that the diverse isolates were produced through sexual reproduction on barberry plants under natural conditions. In addition to P. striiformis f. sp. tritici, sequence analysis of polymerase chain reaction products of the ITS regions and inoculation tests on wheat identified P. graminis (the stem rust pathogen). Our results indicated that P. striiformis f. sp. tritici can infect some Berberis spp. under natural conditions, and the sexual cycle of the fungus may contribute to the diversity of P. striiformis f. sp. tritici in China.