Key Challenges in Plant Pathology in the Next Decade.

Plant diseases significantly impact food security and food safety. It was estimated that food production needs to increase by 50% to feed the projected 9.3 billion people by 2050. Yet, plant pathogens and pests are documented to cause up to 40% yield losses in major crops, including maize, rice, and wheat, resulting in annual worldwide economic losses of approximately US$220 billion. Yield losses due to plant diseases and pests are estimated to be 21.5% (10.1 to 28.1%) in wheat, 30.3% (24.6 to 40.9%) in rice, and 22.6% (19.5 to 41.4%) in maize. In March 2023, The American Phytopathological Society (APS) conducted a survey to identify and rank key challenges in plant pathology in the next decade. Phytopathology subsequently invited papers that address those key challenges in plant pathology, and these were published as a special issue. The key challenges identified include climate change effect on the disease triangle and outbreaks, plant disease resistance mechanisms and its applications, and specific diseases including those caused by Candidatus Liberibacter spp. and Xylella fastidiosa. Additionally, disease detection, natural and man-made disasters, and plant disease control strategies were explored in issue articles. Finally, aspects of open access and how to publish articles to maximize the Findability, Accessibility, Interoperability, and Reuse of digital assets in plant pathology were described. Only by identifying the challenges and tracking progress in developing solutions for them will we be able to resolve the issues in plant pathology and ultimately ensure plant health, food security, and food safety.

Azole resistance mechanisms and population structure of the human pathogen Aspergillus fumigatus on retail plant products.

Aspergillus fumigatus is a ubiquitous saprotroph and human-pathogenic fungus that is life-threatening to the immunocompromised. Triazole-resistant A. fumigatus was found in patients without prior treatment with azoles, leading researchers to conclude that resistance had developed in agricultural environments where azoles are used against plant pathogens. Previous studies have documented azole-resistant A. fumigatus across agricultural environments, but few have looked at retail plant products. Our objectives were to determine if azole-resistant A. fumigatus is prevalent in retail plant products produced in the United States (U.S.), as well as to identify the resistance mechanism(s) and population genetic structure of these isolates. Five hundred twenty-five isolates were collected from retail plant products and screened for azole resistance. Twenty-four isolates collected from compost, soil, flower bulbs, and raw peanuts were pan-azole resistant. These isolates had the TR34/L98H, TR46/Y121F/T289A, G448S, and H147Y cyp51A alleles, all known to underly pan-azole resistance, as well as WT alleles, suggesting that non-cyp51A mechanisms contribute to pan-azole resistance in these isolates. Minimum spanning networks showed two lineages containing isolates with TR alleles or the F46Y/M172V/E427K allele, and discriminant analysis of principle components identified three primary clusters. This is consistent with previous studies detecting three clades of A. fumigatus and identifying pan-azole-resistant isolates with TR alleles in a single clade. We found pan-azole resistance in U.S. retail plant products, particularly compost and flower bulbs, which indicates a risk of exposure to these products for susceptible populations and that highly resistant isolates are likely distributed worldwide on these products.IMPORTANCEAspergillus fumigatus has recently been designated as a critical fungal pathogen by the World Health Organization. It is most deadly to people with compromised immune systems, and with the emergence of antifungal resistance to multiple azole drugs, this disease carries a nearly 100% fatality rate without treatment or if isolates are resistant to the drugs used to treat the disease. It is important to determine the relatedness and origins of resistant A. fumigatus isolates in the environment, including plant-based retail products, so that factors promoting the development and propagation of resistant isolates can be identified.

Detection of azole-resistant Aspergillus fumigatus in the environment from air, plant debris, compost, and soil.

Aspergillus fumigatus is a ubiquitous fungus, a saprophyte of plants, and an opportunistic pathogen of humans. Azole fungicides are used in agriculture to control plant pathogens, and azoles are also used as a first line of treatment for aspergillosis. The continued exposure of A. fumigatus to azoles in the environment has likely led to azole resistance in the clinic where infections result in high levels of mortality. Pan-azole resistance in environmental isolates is most often associated with tandem-repeat (TR) mutations containing 34 or 46 nucleotides in the cyp51A gene. Because the rapid detection of resistance is important for public health, PCR-based techniques have been developed to detect TR mutations in clinical samples. We are interested in identifying agricultural environments conducive to resistance development, but environmental surveillance of resistance has focused on labor-intensive isolation of the fungus followed by screening for resistance. Our goal was to develop assays for the rapid detection of pan-azole-resistant A. fumigatus directly from air, plants, compost, and soil samples. To accomplish this, we optimized DNA extractions for air filters, soil, compost, and plant debris and standardized two nested-PCR assays targeting the TR mutations. Sensitivity and specificity of the assays were tested using A. fumigatus DNA from wild type and TR-based resistant isolates and with soil and air filters spiked with conidia of the same isolates. The nested-PCR assays were sensitive to 5 fg and specific to A. fumigatus without cross-reaction with DNA from other soil microorganisms. Environmental samples from agricultural settings in Georgia, USA were sampled and tested. The TR46 allele was recovered from 30% of samples, including air, soil and plant debris samples from compost, hibiscus and hemp. These assays allow rapid surveillance of resistant isolates directly from environmental samples improving our identification of hotspots of azole-resistant A. fumigatus.

Evidence for the agricultural origin of resistance to multiple antimicrobials in Aspergillus fumigatus, a fungal pathogen of humans.

Pathogen resistance to clinical antimicrobial agents is an urgent problem. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible humans annually. Azoles, which are widely used in both clinical and agricultural settings, are currently the most effective treatment, but resistance to clinical azoles is emerging worldwide. Here, we report the isolation and analysis of azole-sensitive and azole-resistant A. fumigatus from agricultural environments in the southeastern United States (USA) and show that the USA pan-azole-resistant isolates form a clade with pan-azole-resistant isolates from the United Kingdom, the Netherlands, and India. We show that several pan-azole-resistant isolates from agricultural settings in the USA and India also carry alleles with mutations conferring resistance to agricultural fungicides from the benzimidazole (MBC) and quinone outside inhibitor (QoI) classes. We further show that pan-azole-resistant A. fumigatus isolates from patients in clinical settings in the USA, India, and the Netherlands also carry alleles conferring resistance to MBC and QoI agricultural fungicides. The presence of markers for resistance to agricultural-use fungicides in clinical A. fumigatus isolates is strong evidence for an agricultural origin of pan-azole resistance in patients. The presence of multiple fungicide-resistance alleles in agricultural and clinical isolates further suggests that the unique genetics of the pan-azole-resistant clade enables the evolution and/or persistence of antimicrobial resistance mutations leading to the establishment of multifungicide-resistant isolates.

Comparative genomics of host-specialized populations of Corynespora cassiicola causing target spot epidemics in the southeastern United States.

Numerous plant-pathogenic fungi secrete necrotrophic effectors (syn. host-selective toxins) that are important determinants of pathogenicity and virulence in species that have a necrotrophic lifestyle. Corynespora cassiicola is a necrotrophic fungus causing emerging target spot epidemics in the southeastern United States (US). Previous studies revealed that populations of C. cassiicola from cotton, soybean, and tomato are clonal, host specialized and genetically distinct. Additionally, cassiicolin - the necrotrophic effector identified in some C. cassiicola isolates - is an important toxin for virulence on rubber. It is encoded by seven Cas gene variants. Our goal was to conduct comparative genomic analyses to identify variation among putative necrotrophic effector genes and to determine if lack of one of the mating-types explained clonal populations in C. cassiicola causing outbreaks in the southeastern US and the apparent absence of sexual reproduction worldwide. A total of 12 C. cassiicola genomes, with four each from isolates from tomato, soybean, and cotton, were sequenced using an Illumina Next Seq platform. Each genome was assembled de novo, compared with the reference genome from rubber, and searched for known Cas, and other gene clusters with homologs of secondary metabolites. Cas2 and/or Cas6 were present in isolates from soybean in the southeastern US, whereas Cas1 and Cas2 were present in isolates from cotton in the southeastern US. In addition, several toxin genes, including the T-toxin biosynthetic genes were present in all C. cassiicola from cotton, soybean, and tomato. The mating-type locus was identified in all of the sequenced genomes, with the MAT1-1 idiomorph present in all cotton isolates and the rubber isolate, whereas the MAT1-2 idiomorph was present in all soybean isolates. We developed a PCR-based marker for mating-type in C. cassiicola. Both mating types were present in isolates from tomato. Thus, C. cassiicola has both mating-types necessary for sexual reproduction, but the absence of both mating-types within soybean and cotton populations could explain clonality in these populations. Variation in necrotrophic effectors may underlie host specialization and disease emergence of target spot on cotton, soybean, and tomato in the southeastern US.

Azole-resistant Aspergillus fumigatus in the environment: Identifying key reservoirs and hotspots of antifungal resistance.

Aspergillus fumigatus is an opportunistic human pathogen that causes aspergillosis, a spectrum of environmentally acquired respiratory illnesses. It has a cosmopolitan distribution and exists in the environment as a saprotroph on decaying plant matter. Azoles, which target Cyp51A in the ergosterol synthesis pathway, are the primary class of drugs used to treat aspergillosis. Azoles are also used to combat plant pathogenic fungi. Recently, an increasing number of azole-naive patients have presented with pan-azole-resistant strains of A. fumigatus. The TR34/L98H and TR46/Y121F/T289A alleles in the cyp51A gene are the most common ones conferring pan-azole resistance. There is evidence that these mutations arose in agricultural settings; therefore, numerous studies have been conducted to identify azole resistance in environmental A. fumigatus and to determine where resistance is developing in the environment. Here, we summarize the global occurrence of azole-resistant A. fumigatus in the environment based on available literature. Additionally, we have created an interactive world map showing where resistant isolates have been detected and include information on the specific alleles identified, environmental settings, and azole fungicide use. Azole-resistant A. fumigatus has been found on every continent, except for Antarctica, with the highest number of reports from Europe. Developed environments, specifically hospitals and gardens, were the most common settings where azole-resistant A. fumigatus was detected, followed by soils sampled from agricultural settings. The TR34/L98H resistance allele was the most common in all regions except South America where the TR46/Y121F/T289A allele was the most common. A major consideration in interpreting this survey of the literature is sampling bias; regions and environments that have been extensively sampled are more likely to show greater azole resistance even though resistance could be more prevalent in areas that are under-sampled or not sampled at all. Increased surveillance to pinpoint reservoirs, as well as antifungal stewardship, is needed to preserve this class of antifungals for crop protection and human health.

Neofusicoccum caryigenum, a new species causing leaf dieback disease of pecan (Carya illinoinensis).

Neofusicoccum species are endophytes and pathogens of woody hosts and members of the Botryosphaeriaceae. Leaf dieback is a new disease resulting in death of compound leaves and extensive defoliation of pecan trees (Carya illinoinensis) throughout the southeastern United States. Currently, the disease is consistently most severe on trees that are not managed with fungicides for pecan scab. Preliminary observations of the fungus isolated from symptomatic leaves indicated that it was a member of the genus Neofusicoccum. Our objectives were to confirm that this is the causal organism of leaf dieback disease of pecan and to determine whether this disease is caused by a new or previously described species of Neofusicoccum. Morphological observations of pure cultures, conidiomata, conidiogenous cells, and conidia were consistent with members of the genus Neofusicoccum. Using Koch's postulates, we established that Neofusicoccum sp. isolated from symptomatic leaves caused the disease. We sequenced the internal transcribed spacer of the rDNA (ITS), elongation factor 1-α (EF1-α), the second largest subunit of RNA polymerase II (RPB2), and ß-tubulin (TUB2) of 11 isolates collected from Georgia and Texas. Phylogenetic and network analyses of these sequences combined with publicly available sequences of 40 members of the N. parvum-N. ribis species complex and the outgroup N. australe revealed that this fungus is a member of the species complex but is genetically distinct from previously described species. We determined that leaf dieback of pecan is caused by a novel species, named herein N. caryigenum.

Genetic Diversity and Population Structure of Races of Fusarium oxysporum Causing Cotton Wilt.

To better understand the evolution of virulence we are interested in identifying the genetic basis of this trait in pathogenic fungi and in developing tools for the rapid characterization of variation in virulence among populations associated with epidemics. Fusarium oxysporum f. sp. vasinfectum (FOV) is a haploid fungus that causes devastating outbreaks of Fusarium wilt of cotton wherever it is grown. In the United States, six nominal races and eleven genotypes of FOV have been characterized based on the translation elongation factor (EF-1α) gene and intergenic spacer region (IGS), but it is unclear how race or genotype based on these regions relates to population structure or virulence. We used genotyping-by-sequencing to identify SNPs and determine genetic diversity and population structure among 86 diverse FOV isolates. Six individuals of Fusarium oxysporum closely related to FOV were genotyped and included in some analyses. Between 193 and 354 SNPs were identified and included in the analyses depending on the pipeline and filtering criteria used. Phylogenetic trees, minimum spanning networks (MSNs), principal components analysis (PCA), and discriminant analysis of principal components (DAPC) demonstrated that races and genotypes of FOV are generally not structured by EF-1α genotype, nor are they monophyletic groups with the exception of race 4 isolates, which are distinct. Furthermore, DAPC identified between 11 and 14 genetically distinct clusters of FOV, whereas only eight EF-1α genotypes were represented among isolates; suggesting that FOV, especially isolates within the widely distributed and common race 1 genotype, is more genetically diverse than currently recognized.

Invasions of gladiolus rust in North America are caused by a widely-distributed clone of Uromycestransversalis.

Uromyces transversalis, the causal agent of Gladiolus rust, is an invasive plant pathogen in the United States and is regulated as a quarantine pathogen in Europe. The aim of this research was to: (i) determine the origin of introductions of U. transversalis to the United States, (ii) track the movement of genotypes, and (iii) understand the worldwide genetic diversity of the species. To develop molecular markers for genotyping, whole genome sequencing was performed on three isolates collected in the United States. Genomes were assembled de novo and searched for microsatellite regions. Primers were developed and tested on ten isolates from the United States resulting in the identification of 24 polymorphic markers. Among 92 isolates collected from Costa Rica, Mexico, New Zealand, Australia, and the United States there were polymorphisms within isolates with no genotypic diversity detected among isolates; however, missing data among the New Zealand and Australia isolates due to either poor amplification of degraded DNA or null alleles as a result of genetic differences made it difficult to generate conclusions about these populations. The microsatellite loci and flanking regions showed high diversity and two divergent genomes within dikaryotic individuals, yet no diversity among individuals, suggesting that the invasive U. transversalis populations from North America are strictly clonal.

Assessing Fitness Costs and Phenotypic Instability of Fentin Hydroxide and Tebuconazole Resistance in Venturia effusa.

Sensitivity monitoring of Venturia effusa, cause of pecan scab, has revealed insensitivity to fentin hydroxide and tebuconazole, but recent research indicates that the insensitivity to fentin hydroxide is not stable. A study was undertaken to determine if a fitness cost may be responsible for this instability. In this study, experiments were conducted to evaluate fitness components and phenotypic stability of insensitivity of V. effusa to fentin hydroxide and tebuconazole. Conidial production, conidial germination, microcolony growth, sensitivity to osmotic stress, and sensitivity to oxidative stress in the absence of fungicide were compared for isolates with differing sensitivities to both fungicides. Percent conidial germination decreased linearly with increasing fentin hydroxide insensitivity, and microcolony growth on 1.0 mM H2O2 decreased linearly with increasing tebuconazole insensitivity. Stability of resistance was assessed on concentrations of 1.0, 3.0, and 10 µg/ml of both fungicides prior to and after five transfers on non-fungicide-amended medium. Tebuconazole insensitivity was stable after transfers, but fentin hydroxide insensitivity on 1.0 and 3.0 µg/ml decreased significantly after transfers, indicating instability. Here we provide evidence that in V. effusa tebuconazole insensitivity is stable and fentin hydroxide insensitivity is not. These results suggest that fentin-hydroxide-resistant V. effusa isolates have reduced conidial viability compared with sensitive isolates, which may allow the population to regain sensitivity in the absence of this frequently used fungicide.

Fusarium wilt of cotton may commonly result from the interaction of Fusarium oxysporum f. sp. vasinfectum with Belonolaimus longicaudatus.

The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS-12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.The interaction between Fusarium oxysporum f. sp. vasinfectum (Fov) and Meloidogyne incognita (root-knot nematode) resulting in Fusarium wilt (FW) of cotton is well-known. Although Belonolaimus longicaudatus (sting nematode) can also interact with Fov and cause FW, it has long been believed that virtually all of the FW in Georgia is caused by the interaction of Fov with M. incognita. In recent years, FW has been reported more frequently in Georgia, which suggests that something affecting the disease complex may have changed. In 2015 and 2016, a survey of 27 Georgia cotton fields in 10 counties was conducted. At least 10 soil and stem samples per field were collected from individual plants showing symptoms of FW to quantify plant-parasitic nematode levels and identify Fov races. Fov race 1 was identified in all samples in 2015, but one sample also had the LA110 genotype and another sample also had the LA108 genotype. In 2016, all Fov races and genotypes found in 2015 were present, however, MDS­12 and LA127/140 also were found. Meloidogyne incognita was present in 18% of fields in 2015 and 40% in 2016, whereas B. longicaudatus was present in all fields in 2015 and 75% of fields in 2016. Regardless of whether they occurred separately or together, M. incognita and B. longicaudatus were present, respectively, in 18% and 55% of individual samples in 2015 and 40% and 51% in 2016. However, M. incognita without B. longicaudatus was found in 7% of samples in 2015 and 34% in 2016, whereas B. longicaudatus without M. incognita was found in 45% of samples in 2015 and 44% in 2016. We conclude that Fov race 1 continues to be the dominant race in Georgia and many instances of FW in Georgia may be due to Fov interacting with B. longicaudatus and not M. incognita as previously believed.

Differences in fungicide resistance profiles and multiple resistance to a quinone-outside inhibitor (QoI), two succinate dehydrogenase inhibitors (SDHI), and a demethylation inhibitor (DMI) for two Stagonosporopsis species causing gummy stem blight of cucurbits.

BACKGROUND: Gummy stem blight (GSB) is a devastating disease of cucurbits that has been effectively managed with fungicide applications. However, the Stagonosporopsis spp. that cause GSB have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, resistance profiles of unique and clonal genotypes of 113 Stagonosporopsis citrulli and 19 S. caricae isolates to four different fungicides were determined based on in vitro mycelial growth assays and molecular markers based on genes encoding fungicide targets. RESULTS: All 19 S. caricae isolates screened were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All 113 S. citrulli isolates were sensitive to tebuconazole and sensitive to fluopyram, with one exception that was fluopyram-resistant. All isolates of S. citrulli except two were resistant to azoxystrobin. Phenotypic differences in response to boscalid were detected among S. citrulli isolates, but the phenotypes were not associated with multilocus genotypes (MLG) determined by 16 microsatellite loci. Additionally, isolates sharing the same MLG varied by SdhB genotype. A unique mutation of I229V in SdhB, a target of succinate dehydrogenase inhibitor fungicides, was detected for the fluopyram-resistant isolate of S. citrulli. CONCLUSION: Both the lack of association of fungicide resistance profiles with genetic similarity of isolates based on microsatellite loci and the finding that widely distributed MLG varied in fungicide resistance profiles suggest that independent evolutionary events for resistance to boscalid have likely occurred. Frequent genetic recombination within populations may be responsible for resistance to multiple fungicides. This study provides useful information for effectively managing both species of GSB fungi present in the southeastern USA and understanding the evolution of fungicide resistance within populations of plant-pathogenic fungi. © 2019 Society of Chemical Industry.

Clonality and geographic structure of host-specialized populations of Corynespora cassiicola causing emerging target spot epidemics in the southeastern United States.

Corynespora cassiicola is a destructive plant-pathogenic fungus causing widespread target spot epidemics, including outbreaks on cotton, soybean, and tomato in the southeastern United States. Previous studies revealed that populations from the three hosts are genetically distinct and host specialized. Although variation in aggressiveness to cotton and tomato were observed, no genetic diversity was detected within populations sampled from each of these hosts. We aimed to gain a better understanding of the emerging target spot epidemics by developing microsatellite markers for C. cassiicola to assess genetic variation, population structure, and to infer modes of reproduction and mechanisms of dispersal. Two hundred sixty-five isolates from cotton, soybean, tomato, and other host plants were genotyped with 13 microsatellite markers. Genotypic diversity revealed genetic variation within each of the populations collected from different hosts, with the population from cotton dominated by clonal genotypes and showing the least genetic diversity. In addition, C. cassiicola populations on different host species were genetically distinct and structured based on host species. No association between genetic and geographic distances was identified in the tomato populations, and the association in cotton populations was low. However, significant regional geographic structure was detected in the soybean populations of C. cassiicola. These results further support previous findings of introduced host specialized isolates or the evolution of more aggressive strains on each host. The lack of geographic structure suggests that the clones on cotton and tomato spread rapidly, or similar founder populations were established by human-mediated dispersal, and that dispersal is not limited. However, regional geographic structure of populations on soybean suggests limited dispersal among more established populations of C. cassiicola, or genetic differences in founder populations that colonized different geographic areas.

Differences in Sensitivity to a Triazole Fungicide Among Stagonosporopsis Species Causing Gummy Stem Blight of Cucurbits.

Gummy stem blight (GSB) is a destructive disease of cucurbits caused by three closely related Stagonosporopsis species. In the southeastern United States, GSB management relies heavily on triazole fungicides. Our objectives were to determine if resistance to triazoles has developed in populations of GSB fungi in the southeastern United States, and if so, to investigate the molecular basis of resistance. A tebuconazole sensitivity assay was conducted on 303 Stagonosporopsis citrulli and 19 S. caricae isolates collected from the southeastern United States in 2013 and 2014, as well as three S. citrulli, three S. cucurbitacearum, and six S. caricae isolates from other regions or years. Tebuconazole resistance was detected for all 19 S. caricae isolates from the southeastern United States and one S. caricae isolate from Brazil. All S. citrulli and S. cucurbitacearum isolates were sensitive to tebuconazole. For resistant and sensitive isolates of S. caricae, coding and promoter regions of the target gene Cyp51 were sequenced and expression levels of Cyp51 and ScAtrG (an ATP-binding cassette transporter) were measured. Tebuconazole resistance was not associated with mutations within Cyp51, multiple copies of Cyp51, changes in the promoter region, or increased expression of Cyp51 or ScAtrG. Tebuconazole resistance may explain the increase in frequency of S. caricae isolates recovered from GSB-infected cucurbits in Georgia.

Elevated Genetic Diversity in the Emerging Blueberry Pathogen Exobasidium maculosum.

Emerging diseases caused by fungi are increasing at an alarming rate. Exobasidium leaf and fruit spot of blueberry, caused by the fungus Exobasidium maculosum, is an emerging disease that has rapidly increased in prevalence throughout the southeastern USA, severely reducing fruit quality in some plantings. The objectives of this study were to determine the genetic diversity of E. maculosum in the southeastern USA to elucidate the basis of disease emergence and to investigate if populations of E. maculosum are structured by geography, host species, or tissue type. We sequenced three conserved loci from 82 isolates collected from leaves and fruit of rabbiteye blueberry (Vaccinium virgatum), highbush blueberry (V. corymbosum), and southern highbush blueberry (V. corymbosum hybrids) from commercial fields in Georgia and North Carolina, USA, and 6 isolates from lowbush blueberry (V. angustifolium) from Maine, USA, and Nova Scotia, Canada. Populations of E. maculosum from the southeastern USA and from lowbush blueberry in Maine and Nova Scotia are distinct, but do not represent unique species. No difference in genetic structure was detected between different host tissues or among different host species within the southeastern USA; however, differentiation was detected between populations in Georgia and North Carolina. Overall, E. maculosum showed extreme genetic diversity within the conserved loci with 286 segregating sites among the 1,775 sequenced nucleotides and each isolate representing a unique multilocus haplotype. However, 94% of the nucleotide substitutions were silent, so despite the high number of mutations, selective constraints have limited changes to the amino acid sequences of the housekeeping genes. Overall, these results suggest that the emergence of Exobasidium leaf and fruit spot is not due to a recent introduction or host shift, or the recent evolution of aggressive genotypes of E. maculosum, but more likely as a result of an increasing host population or an environmental change.

Evolutionary history and variation in host range of three Stagonosporopsis species causing gummy stem blight of cucurbits.

Recently diverged species may form complexes of morphologically similar, yet genetically distinct lineages that occur in overlapping geographic ranges and niches. Using a multilocus sequencing approach we discovered that gummy stem blight of cucurbits is caused by three genetically distinct species: Stagonosporopsis cucurbitacearum (syn. Didymella bryoniae), Stagonosporopsis citrulli, and Stagonosporopsis caricae, which had previously been considered only a pathogen of papaya. Experiments showed that all three species are pathogenic to cucurbits in the genera Cucurbita, Cucumis, and Citrullus, but only S. caricae is aggressive to papaya. Species tree estimates show that S. citrulli and S. cucurbitacearum are phylogenetically distinct sister species, and that S. caricae is the ancestral lineage. The time estimate for divergence of S. caricae from the ancestor of S. cucurbitacearum and S. citrulli at 72 900 YBP pre-dates domestication of papaya and Cucurbita species in the American tropics. The divergence estimate observed for S. cucurbitacearum and S. citrulli at 10 900 YBP suggests that diversification of Cucurbita species and domestication of gourds and squashes could have driven their divergence. This work highlights the use of molecular systematics and population genetics to elucidate genetic identity among previously unassociated fungi and to understand the patterns of pathogen diversification.

Genome expansion and gene loss in powdery mildew fungi reveal tradeoffs in extreme parasitism.

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

Clonal population structure of the chestnut blight fungus in expanding ranges in southeastern Europe.

Expanding populations are often less genetically diverse at their margins than at the centre of a species' range. Established, older populations of the chestnut blight fungus, Cryphonectria parasitica, are more variable for vegetative compatibility (vc) types than in expanding populations in southeastern Europe where C. parasitica has colonized relatively recently. To test whether vc types represent clones, we genotyped 373 isolates of C. parasitica from southern Italy, Romania, Bulgaria, Macedonia, Greece and Turkey using 11 sequence-characterized amplified region (SCAR) markers. Ten SCAR loci and six vegetative incompatibility (vic) loci were polymorphic in these samples. These populations are clonal by all criteria tested: (i) among 373 isolates, we found only eight multilocus haplotypes, and the same haplotypes were found in multiple countries, sometimes separated in time by as much as 12 years; (ii) the number of haplotypes observed was significantly less than expected under random mating; (iii) populations are in linkage disequilibrium; (iv) the two sets of independent markers, SCARs and vc types, are highly correlated; and (v) sexual structures of C. parasitica were found only in Bulgaria and Romania. One mating type (MAT-1) was found in 98% of the isolates sampled. In contrast, a population in northern Italy, in the central part of the range in Europe, had 12 multilocus haplotypes among 19 isolates. The spread of a few clones could be the result either of founder effect and restricted migration, or these clones have greater fitness than others and spread because they are better adapted to conditions in southeastern Europe.