The role of soil abundance of TxtAB in potato common scab disease severity.

Common scab is an economically costly, soil-borne disease of potato endemic in many potato growing regions. The disease is caused by species of Streptomyces bacteria that produce the phytotoxin Thaxtomin A. The primary disease management tool available to growers is planting resistant cultivars, but no cultivar is fully resistant to common scab and partially resistant cultivars are often not the preferred choice of growers because of agronomic or market considerations. Therefore, growers would benefit from knowledge of the presence and severity of common scab infestations in field soils to make informed planting decisions. We implemented a qPCR diagnostic assay to enable field detection and quantification of all strains of Streptomyces that cause common scab in the United States through amplification of the Thaxtomin A biosynthetic genes. Greenhouse trials confirmed that pathogen abundance was highly correlated with disease severity for five distinct phytopathogenic Streptomyces species, though the degree of disease severity was dependent on the pathogen species. Correlations between the abundance of the Thaxtomin biosynthetic genes from field soil with disease on tubers at field sites across four U.S. states and across two years were not as strong as correlations observed in greenhouse assays. We also developed an effective ddPCR diagnostic assay that also has potential for field quantification of Thaxtomin biosynthetic genes. Further improvement of the PCR assays and added modeling of other environmental factors that impact disease outcome, such as soil composition, can aid growers in making informed planting decisions.

Diverse mobile genetic elements shaped the evolution of Streptomyces virulence.

Mobile genetic elements can innovate bacteria with new traits. In plant pathogenic Streptomyces, frequent and recent acquisition of integrative and conjugative or mobilizable genetic elements is predicted to lead to the emergence of new lineages that gained the capacity to synthesize Thaxtomin, a phytotoxin neccesary for induction of common scab disease on tuber and root crops. Here, we identified components of the Streptomyces-potato pathosystem implicated in virulence and investigated them as a nested and interacting system to reevaluate evolutionary models. We sequenced and analysed genomes of 166 strains isolated from over six decades of sampling primarily from field-grown potatoes. Virulence genes were associated to multiple subtypes of genetic elements differing in mechanisms of transmission and evolutionary histories. Evidence is consistent with few ancient acquisition events followed by recurrent loss or swaps of elements carrying Thaxtomin A-associated genes. Subtypes of another genetic element implicated in virulence are more distributed across Streptomyces. However, neither the subtype classification of genetic elements containing virulence genes nor taxonomic identity was predictive of pathogenicity on potato. Last, findings suggested that phytopathogenic strains are generally endemic to potato fields and some lineages were established by historical spread and further dispersed by few recent transmission events. Results from a hierarchical and system-wide characterization refine our understanding by revealing multiple mechanisms that gene and bacterial dispersion have had on shaping the evolution of a Gram-positive pathogen in agricultural settings.

Mistranslation of the genetic code by a new family of bacterial transfer RNAs.

The correct coupling of amino acids with transfer RNAs (tRNAs) is vital for translating genetic information into functional proteins. Errors during this process lead to mistranslation, where a codon is translated using the wrong amino acid. While unregulated and prolonged mistranslation is often toxic, growing evidence suggests that organisms, from bacteria to humans, can induce and use mistranslation as a mechanism to overcome unfavorable environmental conditions. Most known cases of mistranslation are caused by translation factors with poor substrate specificity or when substrate discrimination is sensitive to molecular changes such as mutations or posttranslational modifications. Here we report two novel families of tRNAs, encoded by bacteria from the Streptomyces and Kitasatospora genera, that adopted dual identities by integrating the anticodons AUU (for Asn) or AGU (for Thr) into the structure of a distinct proline tRNA. These tRNAs are typically encoded next to a full-length or truncated version of a distinct isoform of bacterial-type prolyl-tRNA synthetase. Using two protein reporters, we showed that these tRNAs translate asparagine and threonine codons with proline. Moreover, when expressed in Escherichia coli, the tRNAs cause varying growth defects due to global Asn-to-Pro and Thr-to-Pro mutations. Yet, proteome-wide substitutions of Asn with Pro induced by tRNA expression increased cell tolerance to the antibiotic carbenicillin, indicating that Pro mistranslation can be beneficial under certain conditions. Collectively, our results significantly expand the catalog of organisms known to possess dedicated mistranslation machinery and support the concept that mistranslation is a mechanism for cellular resiliency against environmental stress.

Steroidal glycoalkaloids contribute to anthracnose resistance in Solanum lycopersicum.

Anthracnose is a widespread plant disease caused by various species of the fungal pathogen Colletotrichum. In solanaceous plants such as tomato (Solanum lycopersicum), Colletotrichum infections exhibit a quiescent, asymptomatic state in developing fruit, followed by a transition to necrotrophic infections in ripe fruit. Through analysis of fruit tissue extracts of 95L368, a tomato breeding line that yields fruit with enhanced anthracnose resistance, we identified a role for steroidal glycoalkaloids (SGAs) in anthracnose resistance. The SGA α-tomatine and several of its derivatives accumulated at higher levels, in comparison with fruit of the susceptible tomato cultivar US28, and 95L368 fruit extracts displayed fungistatic activity against Colletotrichum. Correspondingly, ripe and unripe 95L368 fruit displayed enhanced expression of glycoalkaloid metabolic enzyme (GAME) genes, which encode key enzymes in SGA biosynthesis. Metabolomics analysis incorporating recombinant inbred lines generated from 95L368 and US28 yielded strong positive correlations between anthracnose resistance and accumulation of α-tomatine and several derivatives. Lastly, transient silencing of expression of the GAME genes GAME31 and GAME5 in anthracnose-susceptible tomato fruit yielded enhancements to anthracnose resistance. Together, our data support a role for SGAs in anthracnose defense in tomato, with a distinct SGA metabolomic profile conferring resistance to virulent Colletotrichum infections in ripe fruit.

Description of Streptomyces griseiscabiei sp. nov. and reassignment of Streptomyces sp. strain NRRL B-16521 to Streptomyces acidiscabies.

Streptomyces strain NRRL B-2795T (DSM 112329T=NRRL B-2795T) is described as the type strain of Streptomyces griseiscabiei sp. nov. using whole-genome average nucleotide identity and multilocus sequence analyses in addition to phenotypic characterization of carbon source utilization, spore chain morphology, melanin production, salt tolerance, pH tolerance, plant pathogenicity and antibiotic resistance. This strain was previously classified as Streptomyces scabiei but suggested as a potential novel species. A second Streptomyces strain, NRRL B-16521, previously named Streptomyces scabiei, and also previously suggested as a potential novel species, is assigned to Streptomyces acidiscabies based on whole-genome average nucleotide identity. Morphological and biochemical characterizations also support this designation for NRRL B-16521. Both Streptomyces sp. strain NRRL B-2795T and NRRL B-16521 cause common scab on multiple cultivars of potato.

The Phytotoxin Thaxtomin A Is the Primary Virulence Determinant for Scab Disease of Beet, Carrot, and Radish Caused by Streptomyces scabiei.

Several species of Streptomyces cause common scab, a major disease of potato, primarily through the phytotoxic effects of the phytotoxin thaxtomin A. Several phytopathogenic Streptomyces species have also been implicated as the causative agents of scab diseases of taproot crops including beet, carrot, radish, parsnip, and turnip. But the molecular mechanisms employed by Streptomyces to infect these crops is unknown. In this work, we tested the hypothesis that thaxtomin A biosynthesis is also necessary for Streptomyces-caused scab of beet, carrot, radish, and turnip. Thaxtomin A induced plant stunting and cell death of all four of these species. Streptomyces mutants in which the transcriptional regulator of thaxtomin A biosynthesis is disrupted were nonvirulent on all four crops, and complementation of the transcriptional regulator rescued thaxtomin A biosynthesis and plant pathogenicity to wild-type levels. These results demonstrate that thaxtomin A is the primary virulence determinant of scab disease of these other crops.

Genetic Dissection of Early Blight Resistance in Tetraploid Potato.

Early blight, caused by the fungus Alternaria solani, is one of the most economically important diseases of potatoes worldwide. We previously identified a tetraploid potato clone, B0692-4, which is resistant to early blight. To dissect the genetic basis of early blight resistance in this clone, a full-sib tetraploid potato population including 241 progenies was derived from a cross between B0692-4 and a susceptible cultivar, Harley Blackwell, in this study. The population was evaluated for foliage resistance against early blight in field trials in Pennsylvania in 2018 and 2019 and relative area under the disease progress curve (rAUDPC) was determined. The distribution of rAUDPC ranged from 0.016 to 0.679 in 2018, and from 0.017 to 0.554 in 2019. Broad sense heritability for resistance, as measured as rAUDPC, was estimated as 0.66-0.80. The population was also evaluated for foliar maturity in field trials in Maine in 2018 and 2020. A moderate negative correlation between rAUDPC and foliar maturity was detected in both years. A genetic linkage map covering a length of 1469.34 cM with 9124 SNP markers was used for mapping quantitative trait loci (QTL) for rAUDPC and foliar maturity. In 2018, three QTLs for early blight were detected; two of them on chromosome 5 overlapped with QTLs for maturity, and one of them on chromosome 7 was independent of maturity QTL. In 2019, six QTLs for early blight were detected; two QTLs on chromosome 5 overlapped with QTLs for maturity, and the other four QTLs did not overlap with QTLs for maturity. The identification of these QTLs provides new insight into the genetic basis of early blight resistance and may serve as sources for marker-assisted selection for early blight resistance breeding.

Streptomyces caniscabiei sp. nov., which causes potato common scab and is distributed across the world.

Fourteen strains of Streptomyces isolated from scab lesions on potato are described as members of a novel species based on genetic distance, morphological observation and biochemical analyses. Morphological and biochemical characteristics of these strains are distinct from other described phytopathogenic species. Strain NE06-02DT has white aerial mycelium and grey, cylindrical, smooth spores on rectus-flexibilis spore chains. Members of this species group can utilize most of the International Streptomyces Project sugars, utilize melibiose and trehalose, produce melanin, grow on 6-7 % NaCl and pH 5-5.5 media, and are susceptible to oleandomycin (100 µg ml-1), streptomycin (20 µg ml-1) and penicillin G (30 µg ml-1). Though the 16S rRNA gene sequences from several members of this novel species are identical to the Streptomyces bottropensis 16S rRNA gene sequence, whole-genome average nucleotide identity and multi-locus sequence analysis confirm that the strains are members of a novel species. Strains belonging to this novel species have been isolated from the United States, Egypt and China with the earliest known members being isolated in 1961 from common scab lesions of potato in both California, USA, and Maine, USA. The name Streptomyces caniscabiei sp. nov. is proposed for strain NE06-02DT (=DSM111602T=ATCC TSD-236T) and the other members of this novel species group.

A Novel Species-Level Group of Streptomyces Exhibits Variation in Phytopathogenicity Despite Conservation of Virulence Loci.

The genus Streptomyces includes several phytopathogenic species that cause common scab, a devastating disease of tuber and root crops, in particular potato. The diversity of species that cause common scab is unknown. Likewise, the genomic context necessary for bacteria to incite common scab symptom development is not fully characterized. Here, we phenotyped and sequenced the genomes of five strains from a poorly studied Streptomyces lineage. These strains form a new species-level group. When genome sequences within just these five strains are compared, there are no polymorphisms of loci implicated in virulence. Each genome contains the pathogenicity island that encodes for the production of thaxtomin A, a phytotoxin necessary for common scab. Yet, not all sequenced strains produced thaxtomin A. Strains varied from nonpathogenic to highly virulent on two hosts. Unexpectedly, one strain that produced thaxtomin A and was pathogenic on radish was not aggressively pathogenic on potato. Therefore, while thaxtomin A biosynthetic genes and production of thaxtomin A are necessary, they are not sufficient for causing common scab of potato. Additionally, results show that even within a species-level group of Streptomyces strains, there can be aggressively pathogenic and nonpathogenic strains despite conservation of virulence genes.

Multiple immunity-related genes control susceptibility of Arabidopsis thaliana to the parasitic weed Phelipanche aegyptiaca.

Parasitic weeds represent a major threat to agricultural production across the world. Little is known about which host genetic pathways determine compatibility for any host-parasitic plant interaction. We developed a quantitative assay to characterize the growth of the parasitic weed Phelipanche aegyptiaca on 46 mutant lines of the host plant Arabidopsis thaliana to identify host genes that are essential for susceptibility to the parasite. A. thaliana host plants with mutations in genes involved in jasmonic acid biosynthesis/signaling or the negative regulation of plant immunity were less susceptible to P. aegyptiaca parasitization. In contrast, A. thaliana plants with a mutant allele of the putative immunity hub gene Pfd6 were more susceptible to parasitization. Additionally, quantitative PCR revealed that P. aegyptiaca parasitization leads to transcriptional reprograming of several hormone signaling pathways. While most tested A. thaliana lines were fully susceptible to P. aegyptiaca parasitization, this work revealed several host genes essential for full susceptibility or resistance to parasitism. Altering these pathways may be a viable approach for limiting host plant susceptibility to parasitism.

Global spatial risk assessment of sharks under the footprint of fisheries.

Effective ocean management and the conservation of highly migratory species depend on resolving the overlap between animal movements and distributions, and fishing effort. However, this information is lacking at a global scale. Here we show, using a big-data approach that combines satellite-tracked movements of pelagic sharks and global fishing fleets, that 24% of the mean monthly space used by sharks falls under the footprint of pelagic longline fisheries. Space-use hotspots of commercially valuable sharks and of internationally protected species had the highest overlap with longlines (up to 76% and 64%, respectively), and were also associated with significant increases in fishing effort. We conclude that pelagic sharks have limited spatial refuge from current levels of fishing effort in marine areas beyond national jurisdictions (the high seas). Our results demonstrate an urgent need for conservation and management measures at high-seas hotspots of shark space use, and highlight the potential of simultaneous satellite surveillance of megafauna and fishers as a tool for near-real-time, dynamic management.

The Evolution, Ecology, and Mechanisms of Infection by Gram-Positive, Plant-Associated Bacteria.

Gram-positive bacteria are prominent members of plant-associated microbial communities. Although many are hypothesized to be beneficial, some are causative agents of economically important diseases of crop plants. Because the features of Gram-positive bacteria are fundamentally different relative to those of Gram-negative bacteria, the evolution and ecology as well as the mechanisms used to colonize and infect plants also differ. Here, we discuss recent advances in our understanding of Gram-positive, plant-associated bacteria and provide a framework for future research directions on these important plant symbionts.

Molecular Dialog Between Parasitic Plants and Their Hosts.

Parasitic plants steal sugars, water, and other nutrients from host plants through a haustorial connection. Several species of parasitic plants such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.) are major biotic constraints to agricultural production. Parasitic plants are understudied compared with other major classes of plant pathogens, but the recent availability of genomic and transcriptomic data has accelerated the rate of discovery of the molecular mechanisms underpinning plant parasitism. Here, we review the current body of knowledge of how parasitic plants sense host plants, germinate, form parasitic haustorial connections, and suppress host plant immune responses. Additionally, we assess whether parasitic plants fit within the current paradigms used to understand the molecular mechanisms of microbial plant-pathogen interactions. Finally, we discuss challenges facing parasitic plant research and propose the most urgent questions that need to be answered to advance our understanding of plant parasitism.

Cultivar Resistance to Common Scab Disease of Potato Is Dependent on the Pathogen Species.

Common scab of potato is a superficial tuber disease caused by Streptomyces species that produce the phytotoxin thaxtomin. Because common scab development is highly dependent on the effects of this single toxin, the current operating paradigm in common scab pathology is that a potato cultivar resistant to one strain of the common scab pathogen is resistant to all strains. However, cultivar resistance to common scab disease identified in one breeding program is often not durable when tested in other potato breeding programs across the United States. We infected 55 potato cultivar populations with three distinct species of the common scab pathogen and identified cultivars that were resistant or susceptible to all three species and cultivars that had widely varying resistance dependent on the pathogen species. Overall lower virulence was associated with the strain that produces the least thaxtomin. This result showcases several cultivars of potato that are expected to be resistant to the majority of common scab populations but also highlights that many potato cultivars are resistant to only specific species of the pathogen. These results demonstrate that extension specialists and growers must consider their local population of the common scab pathogen when selecting which cultivars to plant for common scab resistance.

Similar bacterial communities on healthy and injured skin of black tip reef sharks.

BACKGROUND: Sharks are in severe global decline due to human exploitation. The additional concern of emerging diseases for this ancient group of fish, however, remains poorly understood. While wild-caught and captive sharks may be susceptible to bacterial and transmissible diseases, recent reports suggest that shark skin may harbor properties that prevent infection, such as a specialized ultrastructure or innate immune properties, possibly related to associated microbial assemblages. To assess whether bacterial community composition differs between visibly healthy and insulted (injured) shark skin, we compared bacterial assemblages of skin covering the gills and the back from 44 wild-caught black-tip reef sharks (Carcharhinus melanopterus) from the Amirante Islands (Seychelles) via 16S rRNA gene amplicon sequencing. RESULTS: Shark skin-associated bacterial communities were diverse (5971 bacterial taxa from 375 families) and dominated by three families of the phylum Proteobacteria typical of marine organisms and environments (Rhodobacteraceae, Alteromonadaceae, Halomonadaceae). Significant differences in bacterial community composition of skin were observed for sharks collected from different sites, but not between healthy or injured skin samples or skin type (gills vs. back). The core microbiome (defined as bacterial taxa present in ≥50% of all samples) consisted of 12 bacterial taxa, which are commonly observed in marine organisms, some of which may be associated with animal host health. CONCLUSION: The conserved bacterial community composition of healthy and injured shark skin samples suggests absence of severe bacterial infections or substantial pathogen propagation upon skin insult. While a mild bacterial infection may have gone undetected, the overall conserved bacterial community implies that bacterial function(s) may be maintained in injured skin. At present, the contribution of bacteria, besides intrinsic animal host factors, to counter skin infection and support rapid wound healing in sharks are unknown. This represents clear knowledge gaps that should be addressed in future work, e.g. by screening for antimicrobial properties of skin-associated bacterial isolates.

Identification of Differentially Methylated Sites with Weak Methylation Effects.

Deoxyribonucleic acid (DNA) methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs) among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM) was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ) twins who have different pain sensitivities-both datasets have weak methylation effects of <1%-show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs) are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same concepts, with the only difference being how methylation information across the genome is summarized. If methylation levels are determined by grouping neighboring cytosine sites, then they are DMRs; if methylation levels are calculated based on single cytosines, they are DMCs.

Herbicide injury induces DNA methylome alterations in Arabidopsis.

The emergence of herbicide-resistant weeds is a major threat facing modern agriculture. Over 470 weedy-plant populations have developed resistance to herbicides. Traditional evolutionary mechanisms are not always sufficient to explain the rapidity with which certain weed populations adapt in response to herbicide exposure. Stress-induced epigenetic changes, such as alterations in DNA methylation, are potential additional adaptive mechanisms for herbicide resistance. We performed methylC sequencing of Arabidopsis thaliana leaves that developed after either mock treatment or two different sub-lethal doses of the herbicide glyphosate, the most-used herbicide in the history of agriculture. The herbicide injury resulted in 9,205 differentially methylated regions (DMRs) across the genome. In total, 5,914 of these DMRs were induced in a dose-dependent manner, wherein the methylation levels were positively correlated to the severity of the herbicide injury, suggesting that plants can modulate the magnitude of methylation changes based on the severity of the stress. Of the 3,680 genes associated with glyphosate-induced DMRs, only 7% were also implicated in methylation changes following biotic or salinity stress. These results demonstrate that plants respond to herbicide stress through changes in methylation patterns that are, in general, dose-sensitive and, at least partially, stress-specific.

Characterizing the Immune-Eliciting Activity of Putative Microbe-Associated Molecular Patterns in Tomato.

Detection of conserved microbe-associated molecular patterns (MAMPs), such as bacterial flagellin, is the first line of active defense in plants against pathogenic invaders. Successful pathogens must subvert this immune response to grow to high population density and cause disease. Flagellin from the bacterial pathogen Pseudomonas was the first identified bacterial MAMP and many species across the plant kingdom have sensitive perception systems for detecting the 22-amino acid epitope known as flg22. Tomato and several other solanaceous plants are also able to independently detect a second epitope of flagellin known as flgII-28. This chapter details four experimental protocols to identify and confirm the immune response-eliciting activity of flagellin and putative MAMPs with focus on the Pseudomonas-tomato pathosystem.