CbCyp51-mediated DMI resistance is modulated by codon bias.

Cercospora leaf spot (CLS), caused by the fungus Cercospora beticola, is the most destructive foliar disease of sugar beet worldwide. Resistance to the sterol demethylation inhibitor (DMI) fungicide tetraconazole has been previously correlated to synonymous and non-synonymous mutations in CbCyp51. Here, we extend these analyses to the DMI fungicides prothioconazole, difenoconazole, and mefentrifluconazole in addition to tetraconazole to confirm whether the synonymous and nonsynonymous mutations at amino acid positions 144 and 170 are associated with resistance to these fungicides. Nearly half of the 593 isolates of C. beticola collected in the Red River Valley of North Dakota and Minnesota in 2021 were resistant to all four DMIs. Another 20% were resistant to tetraconazole and prothioconazole, but sensitive to difenoconazole and mefentrifluconazole. A total of 13% of isolates were sensitive to all DMIs tested. We found five CbCyp51 haplotypes and associated them with phenotypes to the four DMIs. The most predominant haplotype (E170_A/ L144F_C) correlated to resistance to all four DMIs with up to 97.6% accuracy. The second most common haplotype (E170_A/L144) consisted of isolates associated with resistance phenotypes to tetraconazole and prothioconazole while also exhibiting sensitive phenotypes to difenoconazole and mefentrifluconazole with up to 98.4% accuracy. Quantitative PCR did not identify differences in CbCyp51 expression between haplotypes. This study gives an understanding for the importance of codon usage in fungicide resistance and provides crop management acuity for fungicide application decision-making.

Beet Soil-Borne Virus Is a Helper Virus for the Novel Beta vulgaris Satellite Virus 1A.

Sugar beet (Beta vulgaris) is grown in temperate regions around the world as a source of sucrose used for natural sweetening. Sugar beet is susceptible to a number of viral diseases, but identification of the causal agent(s) under field conditions is often difficult due to mixtures of viruses that may be responsible for disease symptoms. In this study, the application of RNAseq to RNA extracted from diseased sugar beet roots obtained from the field and from greenhouse-reared plants grown in soil infested with the virus disease rhizomania (causal agent beet necrotic yellow vein virus; BNYVV) yielded genome-length sequences from BNYVV, as well as beet soil-borne virus (BSBV). The nucleotide identities of the derived consensus sequence of BSBV RNAs ranged from 99.4 to 96.7% (RNA1), 99.3 to 95.3% (RNA2), and 98.3 to 95.9% (RNA3) compared with published BSBV sequences. Based on the BSBV genome consensus sequence, clones of the genomic RNAs 1, 2, and 3 were obtained to produce RNA copies of the genome through in vitro transcription. Capped RNA produced from the clones was infectious when inoculated into leaves of Chenopodium quinoa and B. vulgaris, and extracts from transcript-infected C. quinoa leaves could infect sugar beet seedling roots through a vortex inoculation method. Subsequent exposure of these infected sugar beet seedling roots to aviruliferous Polymyxa betae, the protist vector of both BNYVV and BSBV, confirmed that BSBV derived from the infectious clones could be transmitted by the vector. Co-inoculation of BSBV synthetic transcripts with transcripts of a cloned putative satellite virus designated Beta vulgaris satellite virus 1A (BvSat1A) resulted in the production of lesions on leaves of C. quinoa similar to those produced by inoculation with BSBV alone. Nevertheless, accumulation of genomic RNA and the encoded protein of the satellite virus in co-inoculated leaves was readily detected on Northern and Western blots, respectively, whereas no accumulation of satellite virus products occurred when satellite virus RNA was inoculated alone. The predicted sequence of the detected protein encoded by BvSat1A bears hallmarks of coat proteins of other satellite viruses, and virions of a size consistent with a satellite virus were observed in samples testing positive for the virus. The results demonstrate that BSBV is a helper virus for the novel satellite virus BvSat1A.

First report of Pepper yellow dwarf strain of Beet curly top virus and Spinach curly top Arizona virus in red table beet in Idaho, United States.

In the fall 2021, red table beet plants (Beta vulgaris L. cv 'Eagle') exhibiting stunted growth with shorter petioles were observed at an incidence of 10 to 15 percent in a production field in Payette County, Idaho, United States. In addition to stunting, beet leaves displayed yellowing and mild curling and crumpling, and the roots exhibited hairy root symptoms (sFig.1). To identify potential causal viruses, total RNA was isolated from the leaf and root tissue using RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and subjected to high-throughput sequencing (HTS). Two libraries were prepared, one for the leaf sample and another for the root sample using a ribo-minus TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA). HTS was performed with 150 bp paired-end sequencing on a NovaSeq 6000 (Novogene, Sacramento, CA). Following adapter trimming and removal of host transcripts, 5.9 and 16.2 million reads were obtained from the leaf and root samples, respectively. These reads were de novo assembled using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The assembled leaf sample contigs were aligned to the NCBI non-redundant database to identify contigs matching known viruses. A single contig of 2845 nts that shared 96% coverage and 95.6% sequence identity to the pepper yellow dwarf strain of beet curly top virus (BCTV-PeYD, EU921828; Varsani et al., 2014), and 98% coverage and 98.39% identity with an isolate of BCTV-PeYD (KX529650) from Mexico, was identified in the leaf sample (GenBank Accession OP477336). To validate the HTS detection of BCTV-PeYD, total DNA was isolated from the leaf sample and a 454 bp fragment of the C1 gene (replication-associate protein) was PCR amplified and Sanger sequencing of the amplicon revealed 99.7% identity to the HTS assembled BCTV-PeYD sequence. In addition to the PeYD strain of BCTV, the Worland strain of BCTV (BCTV-Wor) was detected as a single 2930 nt contig with 100% coverage and 97.3% identity to the BCTV-Wor isolate CTS14-015 (KX867045) known to infect sugar beet in Idaho. Of note, there are 11 strains of BCTV and among those, the BCTV-Wor strain induces mild symptoms in sugar beet (Strausbaugh et al., 2017), whereas BCTV-PeYD was found only in pepper from New Mexico. Further, two contigs of 2201 nts and 523 nts were assembled generating a nearly complete genome of spinach curly top Arizona virus (SpCTAV) in the leaf sample with 99% coverage and 99.3% identity (GenBank Accession OQ703946) to the reference genome of SpCTAV (HQ443515; Hernandez-Zepeda et al., 2013). To validate the HTS results, total DNA was isolated from the leaf tissue and PCR amplified a 442 bp fragment that overlaps the V1, V2, and V3 ORFs and its sequence revealed 100% identity with the HTS assembled SpCTAV. The roots sample also showed HTS reads corresponding to BCTV-PeYD and SpCTAV. In addition, beet necrotic yellow vein virus (BNYVV) was detected in the root sample with 30% coverage, but no sequence reads matching to BNYVV was detected in the leaf sample. BNYVV is known to infect sugar beet causing rhizomania (Tamada et al., 1973; Schirmer et al., 2005). To further confirm the BNYVV HTS results, total RNA was extracted separately from the root and leaf tissue, and RT-PCR was performed with primers that were designed to amplify portions of BNYVV RNAs (Weiland et al., 2020). RT-PCR analysis generated the appropriate amplicons with expected sequences corresponding to the RNA-1, RNA-2, RNA-3, and RNA-4 of BNYVV as determined by Sanger sequencing implying BNYVV the causal agent of hairy root symptoms. Similar to observations seen for BNYVV infection in conventional sugar beet varieties, no amplification was detected for BNYVV in the RNA extracted from leaf tissue, indicating that the RT-PCR results are consistent with the HTS analysis. This is the first report of BCTV-PeYD and SpCTAV observed naturally infecting red table beet in Idaho suggesting the geographical expansion of these viruses. The co-existence of BCTV-PeYD and SpCTAV with limited host range needs to be investigated to determine the actual cause of the observed foliar symptoms. This report provides the basis for further research to understand the pathogenic nature of these viruses and their potential threat to red table beet and sugar beet production in Idaho.

Resistance to QoI and DMI Fungicides Does Not Reduce Virulence of C. beticola Isolates in North Central United States.

Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and is managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola, has been reported to be resistant to quinone outside inhibitors (QoIs) and to have reduced sensitive to demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from fields in Foxhome, Minnesota, in 2017. Fitness of these resistant isolates was compared with that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI), and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused as high disease severity as the sensitive ones at 21 DAI. Although QoI- and/or DMI-resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices.

Fungal social influencers: secondary metabolites as a platform for shaping the plant-associated community.

Fungal secondary metabolites (FSMs) are capable of manipulating plant community dynamics by inhibiting or facilitating the establishment of co-habitating organisms. Although production of FSMs is not crucial for survival of the producer, their absence can indirectly impair growth and/or niche competition of these fungi on the plant. The presence of FSMs with no obvious consequence on the fitness of the producer leaves questions regarding ecological impact. This review investigates how fungi employ FSMs as a platform to mediate fungal-fungal, fungal-bacterial and fungal-animal interactions associated with the plant community. We discuss how the biological function of FSMs may indirectly benefit the producer by altering the dynamics of surrounding organisms. We introduce several instances where FSMs influence antagonistic- or alliance-driven interactions. Part of our aim is to decipher the meaning of the FSM 'language' as it is widely noted to impact the surrounding community. Here, we highlight the contribution of FSMs to plant-associated interaction networks that affect the host either broadly or in ways that may have previously been unclear.

Seedborne Cercospora beticola Can Initiate Cercospora Leaf Spot from Sugar Beet (Beta vulgaris) Fruit Tissue.

Cercospora leaf spot (CLS) is a globally important disease of sugar beet (Beta vulgaris) caused by the fungus Cercospora beticola. Long-distance movement of C. beticola has been indirectly evidenced in recent population genetic studies, suggesting potential dispersal via seed. Commercial sugar beet "seed" consists of the reproductive fruit (true seed surrounded by maternal pericarp tissue) coated in artificial pellet material. In this study, we confirmed the presence of viable C. beticola in sugar beet fruit for 10 of 37 tested seed lots. All isolates harbored the G143A mutation associated with quinone outside inhibitor resistance, and 32 of 38 isolates had reduced demethylation inhibitor sensitivity (EC50 > 1 µg/ml). Planting of commercial sugar beet seed demonstrated the ability of seedborne inoculum to initiate CLS in sugar beet. C. beticola DNA was detected in DNA isolated from xylem sap, suggesting the vascular system is used to systemically colonize the host. We established nuclear ribosomal internal transcribed spacer region amplicon sequencing using the MinION platform to detect fungi in sugar beet fruit. Fungal sequences from 19 different genera were identified from 11 different sugar beet seed lots, but Fusarium, Alternaria, and Cercospora were consistently the three most dominant taxa, comprising an average of 93% relative read abundance over 11 seed lots. We also present evidence that C. beticola resides in the pericarp of sugar beet fruit rather than the true seed. The presence of seedborne inoculum should be considered when implementing integrated disease management strategies for CLS of sugar beet in the future.

First report of Tomato bushy stunt virus naturally infecting sugar beet in the United States.

Sugar beet (Beta vulgaris L.) is an important crop grown for its sucrose content used in sugar production around the world. Tomato bushy stunt virus (TBSV) is an RNA virus that belongs to the Tombusvirus genus of the family Tombusviridae (Hearne et al., 1990). The virus was first isolated from tomato, and it is known to infect a wide range of plants (Smith, 1935; Martelli et al., 1988; Hafez et al., 2010). In 1980, a natural infection of TBSV was reported in sugar beet leaves with chlorotic and necrotic ring spots and line pattern symptoms based on serological affinity to TBSV anti-sera in Czechoslovakia (Novak and Lanzova, 1980). In March 2021, sugarbeet plants showing stunted and bushy growth with yellowing and necrotic leaves were observed in a production field in the Imperial Valley of California. Harvested roots exhibited stunted and abnormal growth compared to roots from healthy plants (sFig. 1A). These symptoms prompted a screen for potential infection by TBSV. Root-tissue harvested from the symptomatic sugar beet was initially screened using a TBSV double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA; Agdia, Inc., Elkhart, IN), which reacted positive for TBSV. To obtain the full-length sequence of TBSV and potentially other viruses in the sample, total RNA isolated using the RNeasy Plant Mini Kit (Qiagen, Valencia, CA) from the root-tissue was subjected to high-throughput sequencing (HTS). Libraries were prepared using the TruSeq Stranded Total RNA Library Prep kit (Illumina, San Diego, CA) and sequenced using Illumina NovoSeq 6000 paired-end platform (Novogene, Sacramento, CA). A total of 52 million reads were obtained after removing the adapters and reads mapping to the host genome. These high-quality reads were de novo assembled into 75,891 contigs that are larger than 500 base pairs using the SPAdes assembler (Bankevitch et al., 2012; Prjibelski et al., 2020). The resulting contigs were searched for matching sequences to known viruses using the NCBI non-redundant database. A single contig of 4770 nts representing the full-length genome of TBSV was generated (Accession number OP477335), which showed 100% coverage to previously reported TBSV isolates 'statice' (AJ249740.1) and 'nipplefruit' (AY579432.1) with 92.19% and 91.25% nucleotide sequence identities, respectively, and thus confirming the presence of TBSV in sugar beet root-tissue. However, it showed 74% coverage with only 87% nucleotide identity to a previously reported Lettuce necrotic stunt virus (LNSV) from sugar beet, a tombusvirus that was re-classified as Moroccan pepper virus (MPV) due to high degree (>97%) of sequence identity (Obermeier et al., 2001; Wintermantel and Anchieta, 2012; Wintermantel and Hladky, 2013). The coat protein is conserved within species in tombusvirus, and it plays a significant role by providing serological relationships to tombusvirus taxonomy. The coat protein of TBSV-isolate of this study shared 98.45% and 96.91% identities at amino acid level with TBSV 'nipplefruit' (AY579432.1) and TBSV 'statice' (AJ249740.1) isolates, respectively. In contrast, it showed only 61.56% identity with the coat protein of MPV as shown in the phylogenetic tree indicating that the TBSV-isolate reported here is different from MPV (sFig. 2). To confirm the presence of TBSV, reverse-transcription (RT)-PCR was performed using the total RNA isolated from the root-tissue with primers (VR306: 5'-CGCTCACGAGCCCAGCATCCTTGA-3' and VR297: 5'-ACACCGCCACAGGAGCCATGATTG-3') designed based on the HTS data to amplify a portion of the TBSV genome. Sequencing of the RT-PCR product confirmed the presence of TBSV sequence with 99.1% identity to the TBSV-isolate identified in this study. Further, mechanical inoculation of total RNA isolated from the symptomatic sugar beet roots produced local lesions and systemic necrosis symptoms on the leaves of Chenopodium quinoa (sFig. 1B). Sequencing of the amplicon obtained using RT-PCR with primers VR306 and VR297 confirmed the presence of TBSV in C. quinoa. In addition to TBSV, several viral contigs representing Beet necrotic yellow vein virus were identified in the root-tissue indicating mixed infection in the field. To our knowledge, this is the first report that documents the occurrence of TBSV in sugar beet in the United States. Since TBSV is a soil-borne virus, our findings indicate the need for further studies focused on the frequency and coexistence of the TBSV with BNYVV in sugar beet production fields to understand the disease complexity resulting from potential mixed infections.

Gene cluster conservation provides insight into cercosporin biosynthesis and extends production to the genus Colletotrichum.

Species in the genus Cercospora cause economically devastating diseases in sugar beet, maize, rice, soy bean, and other major food crops. Here, we sequenced the genome of the sugar beet pathogen Cercospora beticola and found it encodes 63 putative secondary metabolite gene clusters, including the cercosporin toxin biosynthesis (CTB) cluster. We show that the CTB gene cluster has experienced multiple duplications and horizontal transfers across a spectrum of plant pathogenic fungi, including the wide-host range Colletotrichum genus as well as the rice pathogen Magnaporthe oryzae Although cercosporin biosynthesis has been thought to rely on an eight-gene CTB cluster, our phylogenomic analysis revealed gene collinearity adjacent to the established cluster in all CTB cluster-harboring species. We demonstrate that the CTB cluster is larger than previously recognized and includes cercosporin facilitator protein, previously shown to be involved with cercosporin autoresistance, and four additional genes required for cercosporin biosynthesis, including the final pathway enzymes that install the unusual cercosporin methylenedioxy bridge. Lastly, we demonstrate production of cercosporin by Colletotrichum fioriniae, the first known cercosporin producer within this agriculturally important genus. Thus, our results provide insight into the intricate evolution and biology of a toxin critical to agriculture and broaden the production of cercosporin to another fungal genus containing many plant pathogens of important crops worldwide.

Rapid Detection of Cercospora beticola in Sugar Beet and Mutations Associated with Fungicide Resistance Using LAMP or Probe-Based qPCR.

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is the most destructive disease of sugar beet worldwide. Although growing CLS-tolerant varieties is helpful, disease management currently requires timely application of fungicides. However, overreliance on fungicides has led to the emergence of fungicide resistance in many C. beticola populations, resulting in multiple epidemics in recent years. Therefore, this study focused on developing a fungicide resistance detection "toolbox" for early detection of C. beticola in sugar beet leaves and mutations associated with different fungicides in the pathogen population. A loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of C. beticola in infected sugar beet leaves. The LAMP primers specific to C. beticola (Cb-LAMP) assay was able to detect C. beticola in inoculated sugar beet leaves as early as 1 day postinoculation. A quinone outside inhibitor (QoI)-LAMP assay was also developed to detect the G143A mutation in cytochrome b associated with QoI resistance in C. beticola. The assay detected the mutation in C. beticola both in vitro and in planta with 100% accuracy. We also developed a probe-based quantitative PCR (qPCR) assay for detecting an E198A mutation in ß-tubulin associated with benzimidazole resistance and a probe-based qPCR assay for detection of mutations in cytochrome P450-dependent sterol 14α-demethylase (Cyp51) associated with resistance to sterol demethylation inhibitor fungicides. The primers and probes used in the assay were highly efficient and precise in differentiating the corresponding fungicide-resistant mutants from sensitive wild-type isolates.

Tools of the crook- infection strategies of fungal plant pathogens.

Fungi represent an ecologically diverse group of microorganisms that includes plant pathogenic species able to cause considerable yield loses in crop production systems worldwide. In order to establish compatible interactions with their hosts, pathogenic fungi rely on the secretion of molecules of diverse nature during host colonization to modulate host physiology, manipulate other environmental factors or provide self-defence. These molecules, collectively known as effectors, are typically small secreted cysteine-rich proteins, but may also comprise secondary metabolites and sRNAs. Here, we discuss the most common strategies that fungal plant pathogens employ to subvert their host plants in order to successfully complete their life cycle and secure the release of abundant viable progeny.

Gene cluster conservation identifies melanin and perylenequinone biosynthesis pathways in multiple plant pathogenic fungi.

Perylenequinones are a family of structurally related polyketide fungal toxins with nearly universal toxicity. These photosensitizing compounds absorb light energy which enables them to generate reactive oxygen species that damage host cells. This potent mechanism serves as an effective weapon for plant pathogens in disease or niche establishment. The sugar beet pathogen Cercospora beticola secretes the perylenequinone cercosporin during infection. We have shown recently that the cercosporin toxin biosynthesis (CTB) gene cluster is present in several other phytopathogenic fungi, prompting the search for biosynthetic gene clusters (BGCs) of structurally similar perylenequinones in other fungi. Here, we report the identification of the elsinochrome and phleichrome BGCs of Elsinoë fawcettii and Cladosporium phlei, respectively, based on gene cluster conservation with the CTB and hypocrellin BGCs. Furthermore, we show that previously reported BGCs for elsinochrome and phleichrome are involved in melanin production. Phylogenetic analysis of the corresponding melanin polyketide synthases (PKSs) and alignment of melanin BGCs revealed high conservation between the established and newly identified C. beticola, E. fawcettii and C. phlei melanin BGCs. Mutagenesis of the identified perylenequinone and melanin PKSs in C. beticola and E. fawcettii coupled with mass spectrometric metabolite analyses confirmed their roles in toxin and melanin production.

Genetic Diversity and Structure in Regional Cercospora beticola Populations from Beta vulgaris subsp. vulgaris Suggest Two Clusters of Separate Origin.

Cercospora leaf spot, caused by Cercospora beticola, is a highly destructive disease of Beta vulgaris subsp. vulgaris worldwide. C. beticola populations are usually characterized by high genetic diversity, but little is known of the relationships among populations from different production regions around the world. This information would be informative of population origin and potential pathways for pathogen movement. For the current study, the genetic diversity, differentiation, and relationships among 948 C. beticola isolates in 28 populations across eight geographic regions were investigated using 12 microsatellite markers. Genotypic diversity, as measured by Simpson's complement index, ranged from 0.18 to 1.00, while pairwise index of differentiation values ranged from 0.02 to 0.42, with the greatest differentiation detected between two New York populations. In these populations, evidence for recent expansion was detected. Assessment of population structure identified two major clusters: the first associated with New York, and the second with Canada, Chile, Eurasia, Hawaii, Michigan, North Dakota, and one population from New York. Inferences of gene flow among these regions suggested that the source for one cluster likely is Eurasia, whereas the source for the other cluster is not known. These results suggest a shared origin of C. beticola populations across regions, except for part of New York, where population divergence has occurred. These findings support the hypothesis that dispersal of C. beticola occurs over long distances.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

Microbial small molecules - weapons of plant subversion.

Covering: up to 2018 Plants live in close association with a myriad of microbes that are generally harmless. However, the minority of microbes that are pathogens can severely impact crop quality and yield, thereby endangering food security. By contrast, beneficial microbes provide plants with important services, such as enhanced nutrient uptake and protection against pests and diseases. Like pathogens, beneficial microbes can modulate host immunity to efficiently colonize the nutrient-rich niches within and around the roots and aerial tissues of a plant, a phenomenon mirroring the establishment of commensal microbes in the human gut. Numerous ingenious mechanisms have been described by which pathogenic and beneficial microbes in the plant microbiome communicate with their host, including the delivery of immune-suppressive effector proteins and the production of phytohormones, toxins and other bioactive molecules. Plants signal to their associated microbes via exudation of photosynthetically fixed carbon sources, quorum-sensing mimicry molecules and selective secondary metabolites such as strigolactones and flavonoids. Molecular communication thus forms an integral part of the establishment of both beneficial and pathogenic plant-microbe relations. Here, we review the current knowledge on microbe-derived small molecules that can act as signalling compounds to stimulate plant growth and health by beneficial microbes on the one hand, but also as weapons for plant invasion by pathogens on the other. As an exemplary case, we used comparative genomics to assess the small molecule biosynthetic capabilities of the Pseudomonas genus; a genus rich in both plant pathogenic and beneficial microbes. We highlight the biosynthetic potential of individual microbial genomes and the population at large, providing evidence for the hypothesis that the distinction between detrimental and beneficial microbes is increasingly fading. Knowledge on the biosynthesis and molecular activity of microbial small molecules will aid in the development of successful biological agents boosting crop resiliency in a sustainable manner and could also provide scientific routes to pathogen inhibition or eradication.

Extensive chromosomal reshuffling drives evolution of virulence in an asexual pathogen.

Sexual recombination drives genetic diversity in eukaryotic genomes and fosters adaptation to novel environmental challenges. Although strictly asexual microorganisms are often considered as evolutionary dead ends, they comprise many devastating plant pathogens. Presently, it remains unknown how such asexual pathogens generate the genetic variation that is required for quick adaptation and evolution in the arms race with their hosts. Here, we show that extensive chromosomal rearrangements in the strictly asexual plant pathogenic fungus Verticillium dahliae establish highly dynamic lineage-specific (LS) genomic regions that act as a source for genetic variation to mediate aggressiveness. We show that such LS regions are greatly enriched for in planta-expressed effector genes encoding secreted proteins that enable host colonization. The LS regions occur at the flanks of chromosomal breakpoints and are enriched for retrotransposons and other repetitive sequence elements. Our results suggest that asexual pathogens may evolve by prompting chromosomal rearrangements, enabling rapid development of novel effector genes. Likely, chromosomal reshuffling can act as a general mechanism for adaptation in asexually propagating organisms.

Mind the gap; seven reasons to close fragmented genome assemblies.

Like other domains of life, research into the biology of filamentous microbes has greatly benefited from the advent of whole-genome sequencing. Next-generation sequencing (NGS) technologies have revolutionized sequencing, making genomic sciences accessible to many academic laboratories including those that study non-model organisms. Thus, hundreds of fungal genomes have been sequenced and are publically available today, although these initiatives have typically yielded considerably fragmented genome assemblies that often lack large contiguous genomic regions. Many important genomic features are contained in intergenic DNA that is often missing in current genome assemblies, and recent studies underscore the significance of non-coding regions and repetitive elements for the life style, adaptability and evolution of many organisms. The study of particular types of genetic elements, such as telomeres, centromeres, repetitive elements, effectors, and clusters of co-regulated genes, but also of phenomena such as structural rearrangements, genome compartmentalization and epigenetics, greatly benefits from having a contiguous and high-quality, preferably even complete and gapless, genome assembly. Here we discuss a number of important reasons to produce gapless, finished, genome assemblies to help answer important biological questions.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

The Genome of the Saprophytic Fungus Verticillium tricorpus Reveals a Complex Effector Repertoire Resembling That of Its Pathogenic Relatives.

Vascular wilts caused by Verticillium spp. are destructive plant diseases affecting hundreds of hosts. Only a few Verticillium spp. are causal agents of vascular wilt diseases, of which V. dahliae is the most notorious pathogen, and several V. dahliae genomes are available. In contrast, V. tricorpus is mainly known as a saprophyte and causal agent of opportunistic infections. Based on a hybrid approach that combines second and third generation sequencing, a near-gapless V. tricorpus genome assembly was obtained. With comparative genomics, we sought to identify genomic features in V. dahliae that confer the ability to cause vascular wilt disease. Unexpectedly, both species encode similar effector repertoires and share a genomic structure with genes encoding secreted proteins clustered in genomic islands. Intriguingly, V. tricorpus contains significantly fewer repetitive elements and an extended spectrum of secreted carbohydrate- active enzymes when compared with V. dahliae. In conclusion, we highlight the technical advances of a hybrid sequencing and assembly approach and show that the saprophyte V. tricorpus shares many hallmark features with the pathogen V. dahliae.

Tomato immune receptor Ve1 recognizes effector of multiple fungal pathogens uncovered by genome and RNA sequencing.

Fungal plant pathogens secrete effector molecules to establish disease on their hosts, and plants in turn use immune receptors to try to intercept these effectors. The tomato immune receptor Ve1 governs resistance to race 1 strains of the soil-borne vascular wilt fungi Verticillium dahliae and Verticillium albo-atrum, but the corresponding Verticillium effector remained unknown thus far. By high-throughput population genome sequencing, a single 50-Kb sequence stretch was identified that only occurs in race 1 strains, and subsequent transcriptome sequencing of Verticillium-infected Nicotiana benthamiana plants revealed only a single highly expressed ORF in this region, designated Ave1 (for Avirulence on Ve1 tomato). Functional analyses confirmed that Ave1 activates Ve1-mediated resistance and demonstrated that Ave1 markedly contributes to fungal virulence, not only on tomato but also on Arabidopsis. Interestingly, Ave1 is homologous to a widespread family of plant natriuretic peptides. Besides plants, homologous proteins were only found in the bacterial plant pathogen Xanthomonas axonopodis and the plant pathogenic fungi Colletotrichum higginsianum, Cercospora beticola, and Fusarium oxysporum f. sp. lycopersici. The distribution of Ave1 homologs, coincident with the presence of Ave1 within a flexible genomic region, strongly suggests that Verticillium acquired Ave1 from plants through horizontal gene transfer. Remarkably, by transient expression we show that also the Ave1 homologs from F. oxysporum and C. beticola can activate Ve1-mediated resistance. In line with this observation, Ve1 was found to mediate resistance toward F. oxysporum in tomato, showing that this immune receptor is involved in resistance against multiple fungal pathogens.

The heterothallic sugarbeet pathogen Cercospora beticola contains exon fragments of both MAT genes that are homogenized by concerted evolution.

Dothideomycetes is one of the most ecologically diverse and economically important classes of fungi. Sexual reproduction in this group is governed by mating type (MAT) genes at the MAT1 locus. Self-sterile (heterothallic) species contain one of two genes at MAT1 (MAT1-1-1 or MAT1-2-1) and only isolates of opposite mating type are sexually compatible. In contrast, self-fertile (homothallic) species contain both MAT genes at MAT1. Knowledge of the reproductive capacities of plant pathogens are of particular interest because recombining populations tend to be more difficult to manage in agricultural settings. In this study, we sequenced MAT1 in the heterothallic Dothideomycete fungus Cercospora beticola to gain insight into the reproductive capabilities of this important plant pathogen. In addition to the expected MAT gene at MAT1, each isolate contained fragments of both MAT1-1-1 and MAT1-2-1 at ostensibly random loci across the genome. When MAT fragments from each locus were manually assembled, they reconstituted MAT1-1-1 and MAT1-2-1 exons with high identity, suggesting a retroposition event occurred in a homothallic ancestor in which both MAT genes were fused. The genome sequences of related taxa revealed that MAT gene fragment pattern of Cercospora zeae-maydis was analogous to C. beticola. In contrast, the genome of more distantly related Mycosphaerella graminicola did not contain MAT fragments. Although fragments occurred in syntenic regions of the C. beticola and C. zeae-maydis genomes, each MAT fragment was more closely related to the intact MAT gene of the same species. Taken together, these data suggest MAT genes fragmented after divergence of M. graminicola from the remaining taxa, and concerted evolution functioned to homogenize MAT fragments and MAT genes in each species.