Hui-Ling Liao

Endemism and functional convergence across the North American soil mycobiome

Significance Microbes control vital ecosystem processes like carbon storage and nutrient recycling. Although megadiversity is a hallmark of microbial communities in nature, we still do not know how microbial diversity determines ecosystem function. We addressed this issue by isolating different geographic and local processes hypothesized to shape fungal community composition and activity in pine forests across the continental United States. Although soil enzyme activity varied across soils according to resource availability, enzyme activity was similar across different fungal communities. These observations indicate that much of fungal diversity plays an equal role in soil biogeochemical cycles. However, soil fungal communities vary dramatically in space, indicating that individual species are endemic to bioregions within the North American continent. Identifying the ecological processes that structure communities and the consequences for ecosystem function is a central goal of ecology. The recognition that fungi, bacteria, and viruses control key ecosystem functions has made microbial communities a major focus of this field. Because many ecological processes are apparent only at particular spatial or temporal scales, a complete understanding of the linkages between microbial community, environment, and function requires analysis across a wide range of scales. Here, we map the biological and functional geography of soil fungi from local to continental scales and show that the principal ecological processes controlling community structure and function operate at different scales. Similar to plants or animals, most soil fungi are endemic to particular bioregions, suggesting that factors operating at large spatial scales, like dispersal limitation or climate, are the first-order determinants of fungal community structure in nature. By contrast, soil extracellular enzyme activity is highly convergent across bioregions and widely differing fungal communities. Instead, soil enzyme activity is correlated with local soil environment and distribution of fungal traits within the community. The lack of structure–function relationships for soil fungal communities at continental scales indicates a high degree of functional redundancy among fungal communities in global biogeochemical cycles.

Genetic isolation between two recently diverged populations of a symbiotic fungus

Fungi are an omnipresent and highly diverse group of organisms, making up a significant part of eukaryotic diversity. Little is currently known about the drivers of fungal population differentiation and subsequent divergence of species, particularly in symbiotic, mycorrhizal fungi. Here, we investigate the population structure and environmental adaptation in Suillus brevipes (Peck) Kuntze, a wind‐dispersed soil fungus that is symbiotic with pine trees. We assembled and annotated the reference genome for Su. brevipes and resequenced the whole genomes of 28 individuals from coastal and montane sites in California. We detected two clearly delineated coast and mountain populations with very low divergence. Genomic divergence was restricted to few regions, including a region of extreme divergence containing a gene encoding for a membrane Na+/H+ exchanger known for enhancing salt tolerance in plants and yeast. Our results are consistent with a very recent split between the montane and coastal Su. brevipes populations, with few small genomic regions under positive selection and a pattern of dispersal and/or establishment limitation. Furthermore, we identify a putatively adaptive gene that motivates further functional analyses to link genotypes and phenotypes and shed light on the genetic basis of adaptive traits.

Genetic Dissection Defines the Roles of Elsinochrome Phytotoxin for Fungal Pathogenesis and Conidiation of the Citrus Pathogen Elsinoë fawcettii

Elsinochrome pigments produced by many phytopathogenic Elsinoë spp. are nonhost-selective toxins which react with oxygen molecules after light activation to produce highly toxic reactive oxygen species. The structures and chemical properties of four derivatives are well known. However, the biological roles of elsinochromes in fungal pathogenesis are poorly understood. Many isolates of Elsinoë fawcettii causing citrus scab are able to produce elsinochromes under axenic conditions. In this article, we report the cloning, expression, and functional characterization of the polyketide synthase-encoding gene, EfPKS1, which we show is required for the production of elsinochromes and fungal pathogenesis. Targeted disruption of EfPKS1 in E.fawcettii completely abrogated elsinochrome production, drastically reduced conidiation, and significantly decreased lesion formation on rough lemon leaves. All mutant phenotypes were restored to the wild type in fungal strains expressing a functional copy of EfPKS1. Accumulation of the EfPKS1 transcript and elsinochromes by a wild-type strain appears to be coordinately regulated by light, nutrients, and pH. The results indicate that the product of EfPKS1 is involved in the biosynthesis of elsinochromes via a fungal polyketide pathway, and that elsinochromes play an important role in fungal pathogenesis.

Gene expression in Citrus sinensis fruit tissues harvested from huanglongbing-infected trees: comparison with girdled fruit

Distribution of viable Candidatus Liberibacter asiaticus (CaLas) in sweet orange fruit and leaves (‘Hamlin’ and ‘Valencia’) and transcriptomic changes associated with huanglongbing (HLB) infection in fruit tissues are reported. Viable CaLas was present in most fruit tissues tested in HLB trees, with the highest titre detected in vascular tissue near the calyx abscission zone. Transcriptomic changes associated with HLB infection were analysed in flavedo (FF), vascular tissue (VT), and juice vesicles (JV) from symptomatic (SY), asymptomatic (AS), and healthy (H) fruit. In SY ‘Hamlin’, HLB altered the expression of more genes in FF and VT than in JV, whereas in SY ‘Valencia’, the number of genes whose expression was changed by HLB was similar in these tissues. The expression of more genes was altered in SY ‘Valencia’ JV than in SY ‘Hamlin’ JV. More genes were also affected in AS ‘Valencia’ FF and VT than in AS ‘Valencia’ JV. Most genes whose expression was changed by HLB were classified as transporters or involved in carbohydrate metabolism. Physiological characteristics of HLB-infected and girdled fruit were compared to differentiate between HLB-specific and carbohydrate metabolism-related symptoms. SY and girdled fruit were smaller than H and ungirdled fruit, respectively, with poor juice quality. However, girdling did not cause misshapen fruit or differential peel coloration. Quantitative PCR analysis indicated that many selected genes changed their expression significantly in SY flavedo but not in girdled flavedo. Mechanisms regulating development of HLB symptoms may lie in the host disease response rather than being a direct consequence of carbohydrate starvation.

Metatranscriptomic analysis of ectomycorrhizal roots reveals genes associated with Piloderma-Pinus symbiosis: improved methodologies for assessing gene expression in situ.

Ectomycorrhizal (EM) fungi form symbiotic associations with plant roots that regulate nutrient exchange between forest plants and soil. Environmental metagenomics approaches that employ next-generation sequencing show great promise for studying EM symbioses; however, metatranscriptomic studies have been constrained by the inherent difficulties associated with isolation and sequencing of RNA from mycorrhizae. Here we apply an optimized method for combined DNA/RNA extraction using field-collected EM fungal-pine root clusters, together with protocols for taxonomic identification of expressed ribosomal RNA, and inference of EM function based on plant and fungal metatranscriptomics. We used transcribed portions of ribosomal RNA genes to identify several transcriptionally dominant fungal taxa associated with loblolly pine including Amphinema, Russula and Piloderma spp. One taxon, Piloderma croceum, has a publically available genome that allowed us to identify patterns of gene content and transcript abundance. Over 1500 abundantly expressed Piloderma genes were detected from mycorrhizal roots, including genes for protein metabolism, cell signalling, electron transport, terpene synthesis and other extracellular activities. In contrast, Piloderma gene encoding an ammonia transporter showed highest transcript abundance in soil samples. Our methodology highlights the potential of metatranscriptomics to identify genes associated with symbiosis and ecosystem function using field-collected samples.

Light controls phospholipase A2α and β gene expression in Citrus sinensis

The low-molecular weight secretory phospholipase A2α (CssPLA2α) and β (CsPLA2β) cloned in this study exhibited diurnal rhythmicity in leaf tissue of Citrus sinensis. Only CssPLA2α displayed distinct diurnal patterns in fruit tissues. CssPLA2α and CsPLA2β diurnal expression exhibited periods of approximately 24 h; CssPLA2α amplitude averaged 990-fold in the leaf blades from field-grown trees, whereas CsPLA2β amplitude averaged 6.4-fold. Diurnal oscillation of CssPLA2α and CsPLA2β gene expression in the growth chamber experiments was markedly dampened 24 h after transfer to continuous light or dark conditions. CssPLA2α and CsPLA2β expressions were redundantly mediated by blue, green, red and red/far-red light, but blue light was a major factor affecting CssPLA2α and CsPLA2β expression. Total and low molecular weight CsPLA2 enzyme activity closely followed diurnal changes in CssPLA2α transcript expression in leaf blades of seedlings treated with low intensity blue light (24 μmol m−2 s−1). Compared with CssPLA2α basal expression, CsPLA2β expression was at least 10-fold higher. Diurnal fluctuation and light regulation of PLA2 gene expression and enzyme activity in citrus leaf and fruit tissues suggests that accompanying diurnal changes in lipophilic second messengers participate in the regulation of physiological processes associated with phospholipase A2 action.

Determination of a transcriptional regulator-like gene involved in biosynthesis of elsinochrome phytotoxin by the citrus scab fungus, Elsinoë fawcettii

Elsinochromes are nonhost-selective, light-activated, polyketide-derived toxins produced by many phytopathogenic Elsinoë species. We recently showed that the polyketide synthase-encoding gene EfPKS1 is essential for elsinochrome biosynthesis in the citrus scab fungus Elsinoë fawcettii. Sequence analysis beyond the EfPKS1 gene identified nine putative ORFs: four genes, designated RDT1, TSF1, PRF1 and ECT1, all encode polypeptides likely to have biosynthetic or efflux functions; five additional genes, OXR1 and EfHP1 to EfHP4, encode hypothetical proteins of unknown function. Northern-blot analysis revealed that expression of these genes in E. fawcettii was not completely correlated with accumulation of elsinochromes under nitrogen limitation, alkaline pH or high concentrations of glucose. Targeted disruption of the TSF1 gene, encoding a putative transcriptional activator, yielded fungal mutants unable to produce elsinochromes, and defective in both conidiation and expression of RDT1, EfPKS1, PRF1 and EfHP1, whereas expression of RDT1, TSF1, PRF1 and ECT1 was nearly abolished in EfPKS1-disrupted mutants. By contrast, expression of OXR1, EfHP2 and EfHP3 was not affected by disrupting either EfPKS1 or TSF1. Taken together, the results indicate that in addition to polyketide synthase, the products of TSF1 and other adjacent genes may also play a crucial role in elsinochrome production.

Continental‐level population differentiation and environmental adaptation in the mushroom Suillus brevipes

Recent advancements in sequencing technology allowed researchers to better address the patterns and mechanisms involved in microbial environmental adaptation at large spatial scales. Here we investigated the genomic basis of adaptation to climate at the continental scale in Suillus brevipes, an ectomycorrhizal fungus symbiotically associated with the roots of pine trees. We used genomic data from 55 individuals in seven locations across North America to perform genome scans to detect signatures of positive selection and assess whether temperature and precipitation were associated with genetic differentiation. We found that S. brevipes exhibited overall strong population differentiation, with potential admixture in Canadian populations. This species also displayed genomic signatures of positive selection as well as genomic sites significantly associated with distinct climatic regimes and abiotic environmental parameters. These genomic regions included genes involved in transmembrane transport of substances and helicase activity potentially involved in cold stress response. Our study sheds light on large‐scale environmental adaptation in fungi by identifying putative adaptive genes and providing a framework to further investigate the genetic basis of fungal adaptation.

Extraction of DNA from orange juice, and detection of bacterium Candidatus Liberibacter asiaticus by real-time PCR.

Orange juice processed from Huanglongbing (HLB) affected fruit is often associated with bitter taste and/or off-flavor. HLB disease in Florida is associated with Candidatus Liberibacter asiaticus (CLas), a phloem-limited bacterium. The current standard to confirm CLas for citrus trees is to take samples from midribs of leaves, which are rich in phloem tissues, and use a quantitative real-time polymerase chain reaction (qPCR) test to detect the 16S rDNA gene of CLas. It is extremely difficult to detect CLas in orange juice because of the low CLas population, high sugar and pectin concentration, low pH, and possible existence of an inhibitor to DNA amplification. The objective of this research was to improve extraction of DNA from orange juice and detection of CLas by qPCR. Homogenization using a sonicator increased DNA yield by 86% in comparison to mortar and pestle extraction. It is difficult to separate DNA from pectin; however, DNA was successfully extracted by treating the juice with pectinase. Application of an elution column successfully removed the unidentified inhibitor to DNA amplification. This work provided a protocol to extract DNA from whole orange juice and detect CLas in HLB-affected fruit.

Characterization of pathogenic variants of Elsinoë fawcettii of citrus implies the presence of new pathotypes and cryptic species in Florida

Elsinoé fawcettii causes scab disease in citrus worldwide. Many E. fawcettii isolates produce the phytotoxin elsinochrome that is required for fungal pathogenesis. We previously observed that most of the field-collected E. fawcettii isolates from Florida produced scab on rough lemon (Citrus jambhiri), grapefruit (Citrus paradisi), and sour orange (Citrus aurantium), belonging to the “Florida Broad Host Range” pathotype. A small portion of the isolates failed to induce scab on sour orange, resembling the “Florida Narrow Host Range” pathotype. Of 71 isolates examined, 5 did not belong to either pathotype. In this study, we further investigated the pathological and genetic diversity of these isolates using three index hosts and molecular analyses. Isolate Ef10 was weakly virulent to grapefruit yet nonpathogenic to others; isolate Ef17 induced strong necrosis on grapefruit and sour orange but not on rough lemon; isolates Ef29 and Ef49 were weakly virulent on rough lemon and grapefruit but nonpathogenic to sour orange. Isolate Ef41 failed to produce detectable levels of elsinochrome and did not affect leaves of any species. This isolate produces smaller hyaline conidia compared with other isolates and is a genotypically distinct strain or subspecies as deduced from restriction fragment length polymorphisms and sequence analysis of both the internal transcribed spacer (ITS) of ribosomal DNA and β-tubulin gene fragments and from random amplified polymorphic DNA fragments. Phylogenetic analysis based on the ITS1 region clearly differentiated isolate Ef41 from the other E. fawcettii isolates. Ef41 also displays very distinct extracellular activities of cell wall degrading enzymes and proteases. The results imply the presence of novel pathogenic variant or cryptic subspecies of E. fawcettii that were not previously described in Florida.