Microbiology and Plant Pathology

Acarospora socialis, the bright cobblestone lichen, is commonly found in southwestern North America. This charismatic yellow lichen is a species of key ecological significance as it is often a pioneer species in new environments. Despite their ecological importance virtually no research has been conducted on the genomics of A. socialis. To address this, we used long-read sequencing to generate the first high-quality draft genome of A. socialis. Lichen thallus tissue was collected from Pinkham Canyon in Joshua Tree National Park, California and deposited in the UC Riverside herbarium under accession #295874. The de novo assembly of the mycobiont partner of the lichen was generated from Pacific Biosciences HiFi long reads and Dovetail Omni-C chromatin capture data. After removing algal and bacterial contigs, the fungal genome was approximately 31.2 Mb consisting of 38 scaffolds with contig and scaffold N50 of 2.4 Mb. The BUSCO completeness score of the assembled genome was 97.5% using the Ascomycota gene set. Information on the genome of A. socialis is important for California conservation purposes given that this lichen is threatened in some places locally by wildfires due to climate change. This reference genome will be used for understanding the genetic diversity, population genomics, and comparative genomics of A. socialis species. Genomic resources for this species will support population and landscape genomics investigations, exploring the use of A. socialis as a bioindicator species for climate change, and in studies of adaptation by comparing populations that occur across aridity gradients in California.

Gene prediction is required to obtain optimal biologically meaningful information from genomic sequences, but automated gene prediction software is imperfect. In this study, we compare the original annotation of the Coccidioides immitis RS genome (the reference strain of C. immitis) to annotations using the Funannotate and Augustus genome prediction pipelines. A total of 25% of the originally predicted genes (denoted CIMG) were not found in either the Funannotate or Augustus predictions. A comparison of Funannotate and Augustus predictions also found overlapping but not identical sets of genes. The predicted genes found only in the original annotation (referred to as CIMG-unique) were less likely to have a meaningful functional annotation and a lower number of orthologs and homologs in other fungi than all CIMG genes predicted by the original annotation. The CIMG-unique genes were also more likely to be lineage-specific and poorly expressed. In addition, the CIMG-unique genes were found in clusters and tended to be more frequently associated with transposable elements than all CIMG-predicted genes. The CIMG-unique genes were more likely to have experimentally determined transcription start sites that were further away from the originally predicted transcription start sites, and experimentally determined initial transcription was less likely to result in stable CIMG-unique transcripts. A sample of CIMG-unique genes that were relatively well expressed and differentially expressed in mycelia and spherules was inspected in a genome browser, and the structure of only about half of them was found to be supported by RNA-seq data. These data suggest that some of the CIMG-unique genes are not authentic gene predictions. Genes that were predicted only by the Funannotate pipeline were also less likely to have a meaningful functional annotation, be shorter, and express less well than all the genes predicted by Funannotate. C. immitis genes predicted by more than one annotation are more likely to have predicted functions, many orthologs and homologs, and be well expressed. Lineage-specific genes are relatively uncommon in this group. These data emphasize the importance and limitations of gene prediction software and suggest that improvements to the annotation of the C. immitis genome should be considered.

Abstract: Background and aims: Interactions between species invasions and climate change have the potential to drive changes in plant communities more than either factor alone. One pathway through which these effects can occur is via changes to the rhizosphere microbial community. Invasive plants can alter these microbial communities affecting natives’ abilities to compete with invaders. At the same time, climate change is leading to more frequent extreme wet and dry events. Understanding the response of plant communities to these combined global change drivers requires a comprehensive approach that assesses the relationship between plant competition and belowground rhizosphere microbial community responses. Methods: Here we use a field experiment in a California grassland with a set of six native annual forbs (i.e., wildflowers) and three invasive annual grasses to test how competition with invasive plants alters both identity and function in the native rhizosphere microbiome, and whether competition between these groups interacts with rainfall to amplify or ameliorate microbial shifts. Results: Metagenomics of rhizosphere communities revealed that drought combined with competition from invaders altered a higher number of functions and families in the native rhizosphere compared to invasive competition alone or drought alone. Watering combined with invasion led to fewer shifts. Conclusion: This suggests invasion-driven shifts in the microbial community may be involved in weakening natives’ ability to cope with climate change, especially drought. Understanding the role of the microbial community under invasion and climate change may be critical to mitigating the negative effects of these interacting global change drivers on native communities. Graphical abstract: Understanding plant community response to global change drivers requires a comprehensive approach that assesses the relationship between plant competition and belowground rhizosphere microbial community responses. (a) In this work, we use a field experiment in a California grassland with a set of native forbs (purple) and invasive grasses (teal) to assess the combined effects of competition and water availability (drought, control, watered) on the rhizosphere microbiome. (b) Drought combined with competition from invaders altered the relative abundance of 36 functions (white) and 22 microbial families (blue) in the native rhizosphere compared to the effects of competition (3 functions, 16 families) or drought alone on natives (not shown: 5 functions, 0 families). (c) Additionally, regardless of watering treatment, invasive grasses sourced more of the taxonomic community in native-invasive mixes and this was exacerbated during drought. Overall, these results suggest invasion-driven shifts in the microbiome may be involved in weakening natives’ ability to cope with climate change, especially drought.

The glassy-winged sharpshooter, Homalodisca vitripennis Germar, is an invasive xylem-feeding leafhopper with a devastating economic impact on California agriculture through transmission of the plant pathogen, Xylella fastidiosa. While studies have focused on X. fastidiosa or known symbionts of H. vitripennis, little work has been done at the scale of the microbiome (the bacterial community) or mycobiome (the fungal community). Here, we characterize the mycobiome and the microbiome of H. vitripennis across Southern California and explore correlations with captivity and host insecticide resistance status. Using high-throughput sequencing of the ribosomal internal transcribed spacer 1 region and the 16S rRNA gene to profile the mycobiome and microbiome, respectively, we found that while the H. vitripennis mycobiome significantly varied across Southern California, the microbiome did not. We also observed a significant difference in both the mycobiome and microbiome between captive and wild H. vitripennis. Finally, we found that the mycobiome, but not the microbiome, was correlated with insecticide resistance status in wild H. vitripennis. This study serves as a foundational look at the H. vitripennis mycobiome and microbiome across Southern California. Future work should explore the putative link between microbes and insecticide resistance status and investigate whether microbial communities should be considered in H. vitripennis management practices. IMPORTANCE The glassy-winged sharpshooter is an invasive leafhopper that feeds on the xylem of plants and transmits the devastating pathogen, Xylella fastidiosa, resulting in significant economic damage to California's agricultural system. While studies have focused on this pathogen or obligate symbionts of the glassy-winged sharpshooter, there is limited knowledge of the bacterial and fungal communities that make up its microbiome and mycobiome. To address this knowledge gap, we explored the composition of the mycobiome and the microbiome of the glassy-winged sharpshooter across Southern California and identified differences associated with geography, captivity, and host insecticide resistance status. Understanding sources of variation in the microbial communities associated with the glassy-winged sharpshooter is an important consideration for developing management strategies to control this invasive insect. This study is a first step toward understanding the role microbes may play in the glassy-winged sharpshooter's resistance to insecticides.

In 2021 and 2022, virus-like symptoms were observed in several cultivars of industrial hemp (Cannabis sativa) in two fields in central Washington, USA. Affected plants had a range of symptoms at different developmental stages, with young plants having severe stunting with shortened internodes and reduced flower mass. Young leaves of infected plants also showed light green to total yellowing, and twirling with twisting margins (Fig. S1). Infections of older plants caused less foliar symptoms that consisted of mosaic, mottling, and mild chlorosis on a few branches with tacoing of older leaves. To assess if symptomatic hemp plants were infected with Beet curly top virus (BCTV) as reported earlier (Giladi et al., 2020; Chiginsky et al., 2021), symptomatic leaves were collected from 38 plants, and the extracted total nucleic acids tested by PCR to amplify a 496-base pair (bp) fragment specific to BCTV coat protein (CP) using primers BCTV2-F 5'-GTGGATCAATTTCCAG-ACAATTATC-3' and BCTV2-R 5'-CCCATAAGAGCCATATCA-AACTTC-3' (Strausbaugh et al. 2008). BCTV was found in 37 of the 38 plants. To further assess the virome of symptomatic hemp plants, total RNA was extracted from symptomatic leaves of four plants using Spectrum total RNA isolation kits (Sigma-Aldrich, St. Louis, MO) and subjected to high-throughput sequencing on an Illumina Novaseq platform in paired-end mode (University of Utah, Salt Lake City, UT). The raw reads (33 to 40 million per sample) were trimmed based on quality and ambiguity and resulting paired-end reads of ≈142 bp length were assembled de novo into a pool of contigs (CLC Genomics Workbench 21, Qiagen Inc.). Virus sequences were identified through BLASTn analysis in GenBank (https://www.ncbi.nlm.nih.gov/blast). One contig of 2,929 nucleotides (nt) obtained from one sample (accession no. OQ068391) showed 99.3% identity with BCTV-Wor strain reported from sugar beet in Idaho (accession no. KX867055 Strausbaugh et al., 2017). Another contig of 1,715 nt from a second sample (accession no. OQ068392) shared 97.3% identity with BCTV-CO strain (accession no. KX867022). Two contig sequences of 2,876 nt (accession no. OQ068388) and 1,399 nt (accession no. OQ068389) obtained from the 3rd and 4th samples showed 97.2% and 98.3% identity, respectively, with Citrus yellow vein-associated virus (CYVaV, accession no. MT893740.1) reported in industrial hemp from Colorado (Chiginsky et al., 2021). Contigs of 256 nt sequence (accession no. OQ068390) obtained from the 3rd and 4th samples also showed 99-100% identity with Hop Latent viroid (HLVd) sequences in GenBank (accessions OK143457 and X07397). These results indicated single infections of BCTV strains and co-infection of CYVaV and HLVd in individual plants. To confirm theagents, symptomatic leaves were collected from 28 randomly selected hemp plants and tested by PCR/RT-PCR using primers specific to BCTV (Strausbaugh et al., 2008), CYVaV (Kwon et al., 2021) and HLVd (Matoušek et al., 2001). Amplicons specific to BCTV (496 bp), CYVaV (658 bp) and HLVd (256 bp) were detected in 28, 25, and 2 samples, respectively. BCTV CP sequences obtained by Sanger sequencing from seven samples showed 100% sequence identity with BCTV-CO and BCTV-Wor strains in six and one samples, respectively. Similarly, sequences of CYVaV- and HLVd-specific amplicons showed 100% identity with corresponding sequences in GenBank. To the best of our knowledge, this is the first report of two strains of BCTV (BCTV-CO and BCTV-Wor), CYVaV, and HLVd infecting industrial hemp in Washington state.

Climate change has exacerbated the effects of abiotic stresses on plant growth and productivity. Drought is one of the most important abiotic stress factors that interfere with plant growth and development. Plant selection and breeding as well as genetic engineering methods used to improve crop drought tolerance are expensive and time consuming. Plants use a myriad of adaptative mechanisms to cope with the adverse effects of drought stress including the association with beneficial microorganisms such as plant growth promoting rhizobacteria (PGPR). Inoculation of plant roots with different PGPR species has been shown to promote drought tolerance through a variety of interconnected physiological, biochemical, molecular, nutritional, metabolic, and cellular processes, which include enhanced plant growth, root elongation, phytohormone production or inhibition, and production of volatile organic compounds. Therefore, plant colonization by PGPR is an eco-friendly agricultural method to improve plant growth and productivity. Notably, the processes regulated and enhanced by PGPR can promote plant growth as well as enhance drought tolerance. This review addresses the current knowledge on how drought stress affects plant growth and development and describes how PGPR can trigger plant drought stress responses at the physiological, morphological, and molecular levels.

During the construction and assembly of the Mars 2020 mission components at two different NASA cleanrooms, several fungal strains were isolated. Based on their colony morphology, two strains that showed yeast-like appearance were further characterized for their phylogenetic position. The species-level classification of these two novel strains, using traditional colony and cell morphology methods combined with the phylogenetic reconstructions using multi-locus sequence analysis (MLSA) based on several gene loci (ITS, LSU, SSU, RPB1, RPB2, CYTB and TEF1), and whole genome sequencing (WGS) was carried out. This polyphasic taxonomic approach supported the conclusion that the two basidiomycetous yeasts belong to hitherto undescribed species. The strain FJI-L2-BK-P3^T, isolated from the Jet Propulsion Laboratory Spacecraft Assembly Facility, was placed in the Naganishia albida clade (Filobasidiales, Tremellomycetes), but is genetically and physiologically different from other members of the clade. Another yeast strain FKI-L6-BK-PAB1^T, isolated from the Kennedy Space Center Payload Hazardous and Servicing Facility, was placed in the genus Cystobasidium (Cystobasidiales, Cystobasidiomycetes) and is distantly related to C. benthicum. Here we propose two novel species with the type strains, Naganishia kalamii sp. nov. (FJI-L2-BK-P3^T = NRRL 64466 = DSM 115730) and Cystobasidium onofrii sp. nov. (FKI-L6-BK-PAB1^T = NRRL 64426 = DSM 114625). The phylogenetic analyses revealed that single gene phylogenies (ITS or LSU) were not conclusive, and MLSA and WGS-based phylogenies were more advantageous for species discrimination in the two genera. The genomic analysis predicted proteins associated with dehydration and desiccation stress-response and the presence of genes that are directly related to osmotolerance and psychrotolerance in both novel yeasts described. Cells of these two newly-described yeasts were exposed to UV-C radiation and compared with N. onofrii, an extremophilic UV-C resistant cold-adapted Alpine yeast. Both novel species were UV resistant, emphasizing the need for collecting and characterizing extremotolerant microbes, including yeasts, to improve microbial reduction techniques used in NASA planetary protection programs.

Species of the phylum Blastocladiomycota, early-diverging zoosporic (flagellated) lineages of fungi, are vastly understudied. This phylum includes the genus Coelomomyces, which consists of more than 80 fungal species that are obligate parasites of arthropods. Known Coelomomyces species lack a complete asexual life cycle, instead surviving through an obligate heterecious alternation of generations life cycle. Despite their global distribution and interesting life cycle, little is known about the genomics of any Coelomomyces species. To address this, we generated three draft-level genomes and annotations for C. lativittatus representing its haploid meiospore, orange gamete, and amber gamete life stages. These draft genome assemblies ranged in size from 5002 to 5799 contigs, with a total length of 19.8-22.8 Mb and a mean of 7416 protein-coding genes. We then demonstrated the utility of these genomes by combining the draft annotations as a reference for analysis of C. lativittatus transcriptomes. We analyzed transcriptomes from across host-associated life stages, including infected larvae and excised mature sporangia from the mosquito Anopheles quadrimaculatus. We identified differentially expressed genes and enriched GO terms both across and within life stages and used these to make hypotheses about C. lativittatus biology. Generally, we found the C. lativittatus transcriptome to be a complex and dynamic expression landscape; GO terms related to metabolism and transport processes were enriched during infection and terms related to dispersal were enriched during sporulation. We further identified five high mobility group (HMG)-box genes in C. lativittatus, three belonging to clades with mating type (MAT) loci from other fungi, as well as four ortholog expansions in C. lativittatus compared with other fungi. The C. lativittatus genomes and transcriptomes reported here are a valuable resource and may be leveraged toward furthering understanding of the biology of these and other early-diverging fungal lineages.

Lichtheimia ornata is an emerging opportunistic Mucorales pathogen that is associated with fatal infections in immunocompromised individuals. While these environmentally acquired infections have rarely been reported to date, cases were noted in a recent analysis of coronavirus disease 2019 (COVID-19)-associated mucormycosis in India. Here, we report the annotated genome sequence of the environmental isolate CBS 291.66.