Pyricularia Are Mostly Host-Specialized with Limited Reciprocal Cross-Infection Between Wheat and Endemic Grasses in Minas Gerais, Brazil.

Wheat blast, caused by Pyricularia oryzae Triticum (PoT), is an emerging threat to global wheat production. The current understanding of the population biology of the pathogen and epidemiology of the disease has been based on phylogenomic studies that compared the wheat blast pathogen with isolates collected from grasses that were invasive to Brazilian wheat fields. In this study, we performed a comprehensive sampling of blast lesions in wheat crops and endemic grasses found in and away from wheat fields in Minas Gerais. A total of 1,368 diseased samples were collected (976 leaves of wheat and grasses and 392 wheat heads), which yielded a working collection of 564 Pyricularia isolates. We show that, contrary to earlier implications, PoT was rarely found on endemic grasses, and, conversely, members of grass-adapted lineages were rarely found on wheat. Instead, most lineages were host-specialized, with constituent isolates usually grouping according to their host of origin. With regard to the dominant role proposed for signalgrass in wheat blast epidemiology, we found only one PoT member in 67 isolates collected from signalgrass grown away from wheat fields and only three members of Urochloa-adapted lineages among hundreds of isolates from wheat. Cross-inoculation assays on wheat and a signalgrass used in pastures (U. brizantha) suggested that the limited cross-infection observed in the field may be due to innate compatibility differences. Whether or not the observed level of cross-infection would be sufficient to provide an inoculum reservoir, or serve as a bridge between wheat growing regions, is questionable and, therefore, deserves further investigation.

A Reevaluation of Phylogenomic Data Reveals that Current Understanding in Wheat Blast Population Biology and Epidemiology Is Obfuscated by Oversights in Population Sampling.

Wheat blast, caused by the Pyricularia oryzae Triticum lineage (PoT), first emerged in Brazil and quickly spread to neighboring countries. Its recent appearance in Bangladesh and Zambia highlights a need to understand the disease's population biology and epidemiology so as to mitigate pandemic outbreaks. Current knowledge is mostly based on characterizations of Brazilian wheat blast isolates and comparison with isolates from non-wheat, endemic grasses. These foregoing studies concluded that the wheat blast population lacks host specificity and, as a result, undergoes extensive gene flow with populations infecting non-wheat hosts. Additionally, based on genetic similarity between wheat blast and isolates infecting Urochloa species, it was proposed that the disease originally emerged via a host jump from this grass and that Urochloa likely plays a central role in wheat blast epidemiology owing to its widespread use as a pasture grass. However, due to inconsistencies with broader phylogenetic studies, we suspected that these seminal studies had not actually sampled the populations normally found on endemic grasses and, instead, had repeatedly isolated members of PoT and the related Lolium pathogen lineage (PoL1). Re-analysis of the Brazilian data as part of a comprehensive, global, phylogenomic dataset that included a small number of South American isolates sampled away from wheat confirmed our suspicion and identified four new P. oryzae lineages on grass hosts. As a result, the conclusions underpinning current understanding in wheat blast's evolution, population biology, and epidemiology are unsubstantiated and could be equivocal.

Recent co-evolution of two pandemic plant diseases in a multi-hybrid swarm.

Most plant pathogens exhibit host specificity but when former barriers to infection break down, new diseases can rapidly emerge. For a number of fungal diseases, there is increasing evidence that hybridization plays a major role in driving host jumps. However, the relative contributions of existing variation versus new mutations in adapting to new host(s) is unclear. Here we reconstruct the evolutionary history of two recently emerged populations of the fungus Pyricularia oryzae that are responsible for two new plant diseases: wheat blast and grey leaf spot of ryegrasses. We provide evidence that wheat blast/grey leaf spot evolved through two distinct mating episodes: the first occurred ~60 years ago, when a fungal individual adapted to Eleusine mated with another individual from Urochloa. Then, about 10 years later, a single progeny from this cross underwent a series of matings with a small number of individuals from three additional host-specialized populations. These matings introduced non-functional alleles of two key host-specificity factors, whose recombination in a multi-hybrid swarm probably facilitated the host jump. We show that very few mutations have arisen since the founding event and a majority are private to individual isolates. Thus, adaptation to the wheat or Lolium hosts appears to have been instantaneous, and driven entirely by selection on repartitioned standing variation, with no obvious role for newly formed mutations.

Seeing double on Cannabis: Haploids and heteroploids of Bipolaris gigantea on hemp and other dicots.

Bipolaris gigantea (= Drechslera gigantea) causes Bipolaris leaf spot (BLS), a devastating and widespread disease on industrial hemp (Cannabis sativa). An investigation of relationships of isolates from hemp and other plants indicated variation in ploidy that has not previously been reported for Bipolaris. Isolates were obtained from BLS lesions on hemp and nearby weeds in 11 Kentucky counties and were similar to each other in morphology and growth characteristics. In total, 23 isolates were analyzed by multilocus phylogenetics, of which seven were also chosen for whole genome shotgun sequencing. Genes for RNA polymerase II subunit 2 (RPB2), translation elongation factor 1-α (TEF1), and mating type (MAT1) indicated that 13 of the isolates were haploid with only a single allele each of RPB2 and TEF1 and either the MAT1-1 or MAT1-2 idiomorph, whereas 10 were apparently "heteroploid" with two alleles each of RPB2 and TEF1 and both MAT1 idiomorphs. Haploids all had identical RPB2 alleles except for a 1-bp difference in two isolates, identical TEF1 alleles, and (if present) identical MAT1-2 alleles. Those alleles were also present in each heteroploid along with either of two related but distinct alleles for each gene. In contrast, haploids and heteroploids shared allelic variation of MAT1-1. In total, four haploid and two heteroploid genotypes were identified. Genome sequence data assembled to 30-32 Mb for each of four haploid isolates, but 10-31 Mb larger sizes for each of three heteroploids depending on sequencing platform and assembly program. The haploids and heteroploids caused similar disease on hemp.

Comparative Transcriptomics Profiling of Perennial Ryegrass Infected with Wild Type or a ΔvelA Epichloë festucae Mutant Reveals Host Processes Underlying Mutualistic versus Antagonistic Interactions.

Epichloë species form bioprotective endophytic symbioses with many cool-season grasses, including agriculturally important forage grasses. Despite its importance, relatively little is known about the molecular details of the interaction and the regulatory genes involved. VelA is a key global regulator in fungal secondary metabolism and development. In previous studies, we showed the requirement of velA for E. festucae to form a mutualistic interaction with Lolium perenne. We showed that VelA regulates the expression of genes encoding proteins involved in membrane transport, fungal cell wall biosynthesis, host cell wall degradation, and secondary metabolism, along with several small-secreted proteins in Epichloë festucae. Here, by a comparative transcriptomics analysis on perennial ryegrass seedlings and mature plants, which are endophyte free or infected with wild type (mutualistic interaction) or mutant ΔvelA E. festucae (antagonistic or incompatible interaction), regulatory effects of the endophytic interaction on perennial ryegrass development was studied. We show that ΔvelA mutant associations influence the expression of genes involved in primary metabolism, secondary metabolism, and response to biotic and abiotic stresses compared with wild type associations, providing an insight into processes defining mutualistic versus antagonistic interactions.

The low abundance of CpG in the SARS-CoV-2 genome is not an evolutionarily signature of ZAP.

The zinc finger antiviral protein (ZAP) is known to restrict viral replication by binding to the CpG rich regions of viral RNA, and subsequently inducing viral RNA degradation. This enzyme has recently been shown to be capable of restricting SARS-CoV-2. These data have led to the hypothesis that the low abundance of CpG in the SARS-CoV-2 genome is due to an evolutionary pressure exerted by the host ZAP. To investigate this hypothesis, we performed a detailed analysis of many coronavirus sequences and ZAP RNA binding preference data. Our analyses showed neither evidence for an evolutionary pressure acting specifically on CpG dinucleotides, nor a link between the activity of ZAP and the low CpG abundance of the SARS-CoV-2 genome.

First report of black root rot (Berkeleyomyces rouxiae) on greenhouse hemp (Cannabis sativa) in Kentucky.

Hemp, Cannabis sativa, was reintroduced to the US in 2014 after 50 years of prohibition. Since then, growers have focused primarily on cannabinoid production using female plants. Thus, most modern hemp has been asexually propagated in greenhouses and transplanted into fields. In December 2019, a commercial transplant producer in Fayette County, Kentucky reported 30% dieback on 'Seagull' hemp. Plants were potted into artificial media (unknown origin) immediately upon removal from the mist bench, and symptoms developed approximately two weeks afterward. Scattered plants developed stunting, yellowing, and wilting symptoms and then died within one week of symptom development. Roots had brown to black lesions distributed along roots that progressed into necrosis. Aleuriospores (chlamydospores) were visible under the dissecting microscope. Root pieces were surface sterilized with 10% household bleach for 2 min and then plated onto cut centers of ~1 cm thick sterilized carrot cross section (Williamson 2014). Resulting colonies were single-spored and maintained on PDA plates on laboratory benchtops (23°C, fluorescent lights). Three isolates were selected for pathogenicity testing and identification. Resulting colonies initially appeared white but darkened to black within two days following the development of aleuriospores (chlamydospores). Aleuriospores were dark brown, formed in chains of 4 to 5, and measured 10.00 to 14.27 × 5.62 to 11.23 µm (n=50). Endoconidia were hyaline, cylindrical, and measured 11.63 to 34.10 × 3.95 to 5.58 µm (n=50). To confirm pathogenicity, soil was washed from three 4-week-old hemp plants (proprietary cultivar) and roots were soaked in a 1x106 spore/mL conidial suspension for 5 minutes. In addition, the roots of three control plants were mock inoculated using sterile water. Inoculated and control plants were transplanted into sterile potting media and maintained in separate trays. Inoculated plants developed leaf yellowing and wilting symptoms 14 dai. Roots examined 21 dai had scattered brown lesions throughout and were colonized by the fungus. Aleuriospores and endoconidia isolated from the symptomatic roots were identical to the inoculum. Control plants did not develop symptoms after 21 dai. For molecular analyses, DNA was extracted from an actively growing (5 d) representative isolate (19FY005) on PDA using Quick-DNA™ Fungal/Bacterial Miniprep Kit (Zymo Research, Irvine, CA). Fragments of commonly used diagnostic loci ITS, ACT, RPBII and Mcm7 were amplified as described by de Beer et al (2014), Fourie et al (2015) and Duong et al (2012). Amplicons were sequenced by Sanger sequencing and the consensus sequences were compared with the NCBI GenBank database by BLASTn. ACT amplicon (OK135163) top hit was B. rouxiae (MF967149.1) with 100% similarity, Mcm7 (OK135165) top hit was B. rouxiae (MF967103.1) with 100% similarity, RPBII (OK135166) top hit was B. rouxiae (MF967194.1) with 99.80% similarity, and ITS (OK135164) top hit was B. rouxiae (MF952402.1) with 99.82% similarity. Berkeleyomyces spp (syn Thielaviopsis sp.) is the causal agent of black root rot in field crops such as cotton and tobacco and in ornamentals such as holly and pansy; it is also a common disease in greenhouse production systems. Introduction of a cryptic sister species in 2017 may suggest that reports of black root rot in many crops may be either B. basicola or B. rouxiae, both species are indistinguishable by morphology (Nel et al. 2017). Although both species have wide host ranges with no host specificity, speciation may be important in terms of pathogenicity, host susceptibility, and other cultural factors. As hemp acreage increases across the US, black root rot may become a more prominent disease in greenhouse transplant production systems and in fields.

Assessing integrated biomarkers of triplefin fish Forsterygion capito inhabiting contaminated marine water - A multivariate approach.

The presence of multiple chemicals in aquatic ecosystems makes evaluation of their real impact on the biota difficult. Integrated biomarkers are therefore needed to evaluate how these chemicals contribute to environmental degradation. The aims of the present study were to evaluate responses to and effects of marine pollution using a series of biomarkers through multivariate analyses. Transcriptional responses of cyp1a (cytochrome P450), mt (metallothionein), vtg (vitellogenin) and cyp19b (cytochrome P450 aromatase); branchial and hepatic histological alterations; and Fulton condition factors (CF) were evaluated, as well as the metals and polycyclic aromatic hydrocarbons present in Forsterygion capito in Auckland, New Zealand. Sites were selected along a contamination gradient: four highly contaminated sites and four less contaminated. Molecular responses with a higher relative expression of the mt and cyp1a genes were detected at a highly contaminated site (Panmure). Several histological lesion types were found in the livers of fish inhabiting both types of sites, but gill lesions were present primarily at highly contaminated sites. In terms of general health status, the lowest CF values were overwhelmingly found in fish from the same site (Panmure). The multivariate approach revealed that telangiectasia and hyperplasia were associated with the presence of chemicals, and these showed negative associations with the CF values, with fish from three highly contaminated sites being most affected. In conclusion, the multivariate approach helped to integrate these biological markers in this blennioid fish, thus providing a more holistic view of the complex chemical mixtures involved. Future studies should implement these analyses.

Human APOBEC3 Variations and Viral Infection.

Human APOBEC3 (apolipoprotein B mRNA-editing catalytic polypeptide-like 3) enzymes are capable of inhibiting a wide range of endogenous and exogenous viruses using deaminase and deaminase-independent mechanisms. These enzymes are essential components of our innate immune system, as evidenced by (a) their strong positive selection and expansion in primates, (b) the evolution of viral counter-defense mechanisms, such as proteasomal degradation mediated by HIV Vif, and (c) hypermutation and inactivation of a large number of integrated HIV-1 proviruses. Numerous APOBEC3 single nucleotide polymorphisms, haplotypes, and splice variants have been identified in humans. Several of these variants have been reported to be associated with differential antiviral immunity. This review focuses on the current knowledge in the field about these natural variations and their roles in infectious diseases.

Telomere Roles in Fungal Genome Evolution and Adaptation.

Telomeres form the ends of linear chromosomes and usually comprise protein complexes that bind to simple repeated sequence motifs that are added to the 3' ends of DNA by the telomerase reverse transcriptase (TERT). One of the primary functions attributed to telomeres is to solve the "end-replication problem" which, if left unaddressed, would cause gradual, inexorable attrition of sequences from the chromosome ends and, eventually, loss of viability. Telomere-binding proteins also protect the chromosome from 5' to 3' exonuclease action, and disguise the chromosome ends from the double-strand break repair machinery whose illegitimate action potentially generates catastrophic chromosome aberrations. Telomeres are of special interest in the blast fungus, Pyricularia, because the adjacent regions are enriched in genes controlling interactions with host plants, and the chromosome ends show enhanced polymorphism and genetic instability. Previously, we showed that telomere instability in some P. oryzae strains is caused by novel retrotransposons (MoTeRs) that insert in telomere repeats, generating interstitial telomere sequences that drive frequent, break-induced rearrangements. Here, we sought to gain further insight on telomeric involvement in shaping Pyricularia genome architecture by characterizing sequence polymorphisms at chromosome ends, and surrounding internalized MoTeR loci (relics) and interstitial telomere repeats. This provided evidence that telomere dynamics have played historical, and likely ongoing, roles in shaping the Pyricularia genome. We further demonstrate that even telomeres lacking MoTeR insertions are poorly preserved, such that the telomere-adjacent sequences exhibit frequent presence/absence polymorphism, as well as exchanges with the genome interior. Using TERT knockout experiments, we characterized chromosomal responses to failed telomere maintenance which suggested that much of the MoTeR relic-/interstitial telomere-associated polymorphism could be driven by compromised telomere function. Finally, we describe three possible examples of a phenomenon known as "Adaptive Telomere Failure," where spontaneous losses of telomere maintenance drive rapid accumulation of sequence polymorphism with possible adaptive advantages. Together, our data suggest that telomere maintenance is frequently compromised in Pyricularia but the chromosome alterations resulting from telomere failure are not as catastrophic as prior research would predict, and may, in fact, be potent drivers of adaptive polymorphism.

Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae.

Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an incision in the meristem, as previously described by Latch and Christensen (1985). In recent years, this method has been broadly used to study this interaction at the molecular level using different Epichloë festucae mutants that can cause incompatible interactions. We have developed and adapted methods to study four of the most important host plant responses to infection, including cell death, callose deposition, lignin production, and hydrogen peroxide (H2O2) production, which are useful in defining the host response to infection at a very early time point.

Bio-indicators in cadmium toxicity: Role of HSP27 and HSP70.

Heat shock proteins (HSPs) are a family of proteins that are expressed by cells in reply to stressors. The changes in concentration of HSPs could be utilized as a bio-indicator of oxidative stress caused by heavy metal. Exposure to the different heavy metals may induce or reduce the expression of different HSPs. The exposure to cadmium ion (Cd2+) could increase HSP70 and HSP27 over 2- to 10-fold or even more. The in vitro and in vivo models indicate that the HSP70 family is more sensitive to Cd intoxication than other HSPs. The analyses of other HSPs along with HSP70, especially HSP27, could also be useful to obtain more accurate results. In this regard, this review focuses on examining the literature to bold the futuristic uses of HSPs as bio-indicators in the initial assessment of Cd exposure risks in defined environments.

Reemergence of Septoria Leaf Spot Caused by Septoria cannabis on Hemp in Kentucky.

Hemp reemerged in 2014 after being illegal for over 50 years and restricted for 90 years. Today, hemp is primarily grown for production of cannabidiol (CBD), with limited acreage dedicated to fiber. One of the most frequent and destructive leaf spot diseases of hemp cultivars grown for CBD is Septoria leaf spot. Symptoms are mostly observed in lower leaves and inner canopy. Leaf spots begin as small, irregular, brown to gray spots that rapidly expand to about 5.0 to 7.5 mm in diameter. Pycnidia are scattered, round, dark brown to black in color, and measure 38.8 to 145.0 µm in diameter. Conidia are hyaline, curved but occasionally straight, pointed at the apex, and contain 3 to 4 septa. Morphological characteristics were similar to those reported for Septoria cannabis. Gene sequences from seven diagnostic loci (elongation factor, ß-tubulin, RNA polymerase II, large subunit, internal transcribed spacer, actin, and calmodulin) did not match any published accessions. There are no published sequences for S. cannabis available for comparison. Phylogenetic analysis of concatenated sequences showed that isolates from hemp grouped separately from other Septoria spp. Similarity of morphological characteristics and lack of sequence data matching other Septoria spp. led to the conclusion that isolates collected from hemp in Kentucky are S. cannabis. This new information will serve as an update for Septoria leaf spot diagnostics, especially as hemp acreage continues to increase across the United States.

Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus.

The fungus Magnaporthe oryzae causes devastating diseases of crops, including rice and wheat, and in various grasses. Strains from ryegrasses have highly unstable chromosome ends that undergo frequent rearrangements, and this has been associated with the presence of retrotransposons (Magnaporthe oryzae Telomeric Retrotransposons-MoTeRs) inserted in the telomeres. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, mapping, and sequencing of parental and novel telomeric restriction fragments (TRFs), along with MinION sequencing of genomic DNA allowed us to document the precise molecular alterations underlying 109 newly-formed TRFs. These included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by 'plain' telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; MoTeR-independent expansion and contraction of subtelomeric tandem repeats; and a variety of rearrangements initiated through breaks in interstitial telomere tracts that are generated during MoTeR integration. Overall, we estimate that alterations occurred in approximately sixty percent of chromosomes (one in three telomeres) analyzed. Most importantly, we describe an entirely new mechanism by which transposons can promote genomic alterations at exceptionally high frequencies, and in a manner that can promote genome evolution while minimizing collateral damage to overall chromosome architecture and function.

Effect of dietary inclusion of lecithin with choline on physiological stress of serum cholesterol fractions and enzymes, abdominal fat, growth performance, and mortality parameters of broiler chickens.

A total of 270 one-d-old Ross 308 broiler chicks were randomly allotted to 9 experimental diets (3 replicates of 10 birds each), including three types of supplemental lipotropic factors (control, 0.1% or 0.2% choline and 0.5% or 1% lecithin) in a 3 × 3 factorial arrangement. Supplementation of lecithin improved FCR values during 1-21 days of age. Although no differences were noticed for mortality index among different diets, the group supplemented with a combination of choline (0.1) and lecithin (0.5) showed the highest (P < 0.0001) production index. Choline (0.1% or 0.2%) significantly decreased serum total cholesterol by 11%, triglycerides by 21%, low-density lipoprotein (LDL) by 20%, and very low-density lipoprotein (VLDL) by 20%, while increased the glucose and high-density lipoprotein (HDL) values by 11% and 6%, respectively. On the other hand, lecithin significantly increased glucose, total cholesterol, triglycerides, HDL, LDL and VLDL by 4%, 9%, 7%, 24%, and 25%, respectively. Choline supplementation decreased the aspartate amino transferase (AST), alanine amino transferase (ALT), and alkaline phosphatase (AP); however, the lecithin addition increased their respective proportions. This study concluded that the combinations of 0.1% choline and 0.5% lecithin is the best among all other treatments because of the highest production index and least mortality.

VelA and LaeA are Key Regulators of Epichloë festucae Transcriptomic Response during Symbiosis with Perennial Ryegrass.

VelA (or VeA) is a key global regulator in fungal secondary metabolism and development which we previously showed is required during the symbiotic interaction of Epichloë festucae with perennial ryegrass. In this study, comparative transcriptomic analyses of ∆velA mutant compared to wild-type E. festucae, under three different conditions (in culture, infected seedlings, and infected mature plants), were performed to investigate the impact of VelA on E. festucae transcriptome. These comparative transcriptomic studies showed that VelA regulates the expression of genes encoding proteins involved in membrane transport, fungal cell wall biosynthesis, host cell wall degradation, and secondary metabolism, along with a number of small secreted proteins and a large number of proteins with no predictable functions. In addition, these results were compared with previous transcriptomic experiments that studied the impact of LaeA, another key global regulator of secondary metabolism and development that we have shown is important for E. festucae-perennial ryegrass interaction. The results showed that although VelA and LaeA regulate a subset of E. festucae genes in a similar manner, they also regulated many other genes independently of each other suggesting specialised roles.

Genetic Manipulation of Chlamydia trachomatis: Chromosomal Deletions.

Progress in understanding molecular mechanisms contributing to chlamydial pathogenesis has been greatly facilitated by recent advances in genetic manipulation of C. trachomatis. Valuable approaches such as random, chemically induced mutagenesis or targeted, insertion-based gene disruption have led to significant discoveries. We describe herein a technique for generating definitive null strains via complete deletion of chromosomal genes in C. trachomatis. Fluorescence-reported allelic exchange mutagenesis (FRAEM), using the suicide vector pSUmC, enables targeted deletion of desired chromosomal DNA. The protocol provided here describes steps required to produce transformation competent chlamydiae, generate a specific allelic exchange plasmid construct, carry out mutagenesis, and isolate clonal populations of resulting mutant strains.

Transformation of Chlamydia: current approaches and impact on our understanding of chlamydial infection biology.

The intonation "The king is dead, long live the king" aptly describes the state of Chlamydia research. Genetic-based approaches are rapidly replacing correlative strategies to provide new insights. We describe how current transformation technologies are enhancing progress in understanding Chlamydia infection biology and present key opportunities for further development.