A Bacillus velezensis strain shows antimicrobial activity against soilborne and foliar fungi and oomycetes.

Biological control uses naturally occurring antagonists such as bacteria or fungi for environmentally friendly control of plant pathogens. Bacillus spp. have been used for biocontrol of numerous plant and insect pests and are well-known to synthesize a variety of bioactive secondary metabolites. We hypothesized that bacteria isolated from agricultural soil would be effective antagonists of soilborne fungal pathogens. Here, we show that the Delaware soil isolate Bacillus velezensis strain S4 has in vitro activity against soilborne and foliar plant pathogenic fungi, including two with a large host range, and one oomycete. Further, this strain shows putative protease and cellulase activity, consistent with our prior finding that the genome of this organism is highly enriched in antifungal and antimicrobial biosynthetic gene clusters. We demonstrate that this bacterium causes changes to the fungal and oomycete hyphae at the inhibition zone, with some of the hyphae forming bubble-like structures and irregular branching. We tested strain S4 against Magnaporthe oryzae spores, which typically form germ tubes and penetration structures called appressoria, on the surface of the leaf. Our results suggest that after 12 hours of incubation with the bacterium, fungal spores form germ tubes, but instead of producing appressoria, they appear to form rounded, bubble-like structures. Future work will investigate whether a single antifungal molecule induces all these effects, or if they are the result of a combination of bacterially produced antimicrobials.

Visualizing Early Infection Sites of Rice Blast Disease (Magnaporthe oryzae) on Barley (Hordeum vulgare) Using a Basic Microscope and a Smartphone.

Understanding how plants and pathogens interact, and whether that interaction culminates in defense or disease, is required to develop stronger and more sustainable strategies for plant health. Advances in methods that more effectively image plant-pathogen samples during infection and colonization have yielded tools such as the rice leaf sheath assay, which has been useful in monitoring infection and early colonization events between rice and the fungal pathogen, Magnaporthe oryzae. This hemi-biotrophic pathogen causes severe disease loss in rice and related monocots, including millet, rye, barley, and more recently, wheat. The leaf sheath assay, when performed correctly, yields an optically clear plant section, several layers thick, which allows researchers to perform live-cell imaging during pathogen attack or generate fixed samples stained for specific features. Detailed cellular investigations into the barley-M. oryzae interaction have lagged behind those of the rice host, in spite of the growing importance of this grain as a food source for animals and humans and as fermented beverages. Reported here is the development of a barley leaf sheath assay for intricate studies of M. oryzae interactions during the first 48 h post-inoculation. The leaf sheath assay, regardless of which species is being studied, is delicate; provided is a protocol that covers everything, from barley growth conditions and obtaining a leaf sheath, to inoculation, incubation, and imaging of the pathogen on plant leaves. This protocol can be optimized for high-throughput screening using something as simple as a smartphone for imaging purposes.

Genome assembly of a Mesoamerican derived variety of lima bean: a foundational cultivar in the Mid-Atlantic USA.

Lima bean, Phaseolus lunatus, is closely related to common bean and is high in fiber and protein, with a low glycemic index. Lima bean is widely grown in the state of Delaware, where late summer and early fall weather are conducive to pod production. The same weather conditions also promote diseases such as pod rot and downy mildew, the latter of which has caused previous epidemics. A better understanding of the genes underlying resistance to this and other pathogens is needed to keep this industry thriving in the region. Our current study sought to sequence, assemble, and annotate a commercially available cultivar called Bridgeton, which could then serve as a reference genome, a basis of comparison to other Phaseolus taxa, and a resource for the identification of potential resistance genes. Combined efforts of sequencing, linkage, and comparative analysis resulted in a 623 Mb annotated assembly for lima bean, as well as a better understanding of an evolutionarily dynamic resistance locus in legumes.

Combined effects of arsenic and Magnaporthe oryzae on rice and alleviation by silicon.

While the impacts of arsenic (As) and Magnaporthe oryzae on rice have been well-studied, a dearth of knowledge exists on how rice responds to their combined stress. Moreover, increasing exogenous silicon (Si) can alleviate M. oryzae infection and As uptake, but how increasing exogenous Si affects the combined stress of M. oryzae and As is unknown. We grew three cultivars of rice that varied in their susceptibility to As and M. oryzae under low (50 µM, SiL) and high (1500 µM, SiH) Si with and without As (4 µM, 80/20 As (III)/As(V)) and with or without M. oryzae infection and examined the impacts of treatments on plant As and Si concentrations, severity of disease by M. oryzae, and stress via targeted gene expression. SiH treatments generally decreased shoot As concentrations by 20-70% compared to SiL treatments depending on cultivar and M. oryzae exposure. There was no effect of Si or As treatments on percent of leaf diseased in the As-tolerant cultivar M206, but in the As-sensitive cultivar IR66, SiH treatment decreased percent of leaf diseased in the absence of As and had no impact when As was present. In the M. oryzae-susceptible Sariceltik, plants receiving SiH had significantly fewer lesions than those receiving SiL and plants with the fewest lesions were in the SiH + As treatments. Plants that were exposed to As + M. oryzae were the most stressed when grown under SiL, but this stress response was lowered by SiH treatments. A separate pathogenicity assay with Sariceltik showed that in contrast to our hypothesis, As exposure decreased lesion growth, particularly under SiH treatments, and lessened the impact of M. oryzae on rice. These results suggest that rice grown under replete Si will be able to withstand combined stressors of M. oryzae and As, but will be highly stressed under Si deficient scenarios.

Complete Genome Sequence of Bacillus velezensis Strain S4, Isolated from Biochar-Treated Soil.

Here, we report the complete genome sequence of Bacillus velezensis strain S4, which was isolated from biochar-amended agricultural soil collected in Smyrna, Delaware. The genome is 4.07 Mbp, encodes 3,918 predicted proteins, and has a GC content of 46.4%.

Genome sequencing and transcript analysis of Hemileia vastatrix reveal expression dynamics of candidate effectors dependent on host compatibility.

Coffee leaf rust caused by the fungus Hemileia vastatrix is one of the most important leaf diseases of coffee plantations worldwide. Current knowledge of the H. vastatrix genome is limited and only a small fraction of the total fungal secretome has been identified. In order to obtain a more comprehensive understanding of its secretome, we aimed to sequence and assemble the entire H. vastatrix genome using two next-generation sequencing platforms and a hybrid assembly strategy. This resulted in a 547 Mb genome of H. vastatrix race XXXIII (Hv33), with 13,364 predicted genes that encode 13,034 putative proteins with transcriptomic support. Based on this proteome, 615 proteins contain putative secretion peptides, and lack transmembrane domains. From this putative secretome, 111 proteins were identified as candidate effectors (EHv33) unique to H. vastatrix, and a subset consisting of 17 EHv33 genes was selected for a temporal gene expression analysis during infection. Five genes were significantly induced early during an incompatible interaction, indicating their potential role as pre-haustorial effectors possibly recognized by the resistant coffee genotype. Another nine genes were significantly induced after haustorium formation in the compatible interaction. Overall, we suggest that this fungus is able to selectively mount its survival strategy with effectors that depend on the host genotype involved in the infection process.

Complete Genome Sequence of Spiroplasma phoeniceum Strain P40T, a Plant Pathogen Isolated from Diseased Plants of Madagascar Periwinkle [Catharanthus roseus (L.) G. Don].

The phytopathogen Spiroplasma phoeniceum was isolated from diseased plants of Madagascar periwinkle [Catharanthus roseus (L.) G. Don]. Here, we report the nucleotide sequence of the 1,791,576-bp circular chromosome and three plasmids of strain P40T This information serves as a resource for comparative analyses of spiroplasmal adaptations to diverse ecological niches.

Characterizing Small RNAs in Filamentous Fungi Using the Rice Blast Fungus, Magnaporthe oryzae, as an Example.

The goal of this chapter is to provide a framework of sequential steps for small RNA (sRNA) analysis in filamentous fungi. Here, we present protocols for (1) comparative analysis of sRNAs in different conditions, (2) comparisons of sRNA libraries to RNAseq data and (3) identification and analysis of methylguanosine-capped and polyadenylated sRNAs (CPA-sRNAs). This species of small RNA is particularly interesting in Magnaporthe oryzae, as they map to transcription start and end sites of protein-coding genes. While we do not provide specific command lines for scripts, we provide a general framework for steps needed to carry out all three types of analyses, including relevant references, websites and free online tools. Screenshots are provided from our own customized interface using M. oryzae as an example, to assist the reader in visualizing many of the steps.

Small RNA Functions Are Required for Growth and Development of Magnaporthe oryzae.

RNA interference (RNAi) is conserved in eukaryotic organisms, and it has been well studied in many animal and plant species and some fungal species, yet it is not well studied in fungal plant pathogens. In the rice blast fungus Magnaporthe oryzae, we examined small RNA (sRNA) and their biogenesis in the context of growth and pathogenicity. Through genetic and genomic analyses, we demonstrate that loss of a single gene encoding Dicer, RNA-dependent RNA polymerase, or Argonaute reduces sRNA levels. These three proteins are required for the biogenesis of sRNA-matching genome-wide regions (coding regions, repeats, and intergenic regions). The loss of one Argonaute reduced both sRNA and fungal virulence on barley leaves. Transcriptome analysis of multiple mutants revealed that sRNA play an important role in transcriptional regulation of repeats and intergenic regions in M. oryzae. Together, these data support that M. oryzae sRNA regulate developmental processes including, fungal growth and virulence.

Marker-Assisted Molecular Profiling, Deletion Mutant Analysis, and RNA-Seq Reveal a Disease Resistance Cluster Associated with Uromyces appendiculatus Infection in Common Bean Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L.) is an important legume, useful for its high protein and dietary fiber. The fungal pathogen Uromyces appendiculatus (Pers.) Unger can cause major loss in susceptible varieties of the common bean. The Ur-3 locus provides race specific resistance to virulent strains or races of the bean rust pathogen along with Crg, (Complements resistance gene), which is required for Ur-3-mediated rust resistance. In this study, we inoculated two common bean genotypes (resistant "Sierra" and susceptible crg) with rust race 53 of U. appendiculatus, isolated leaf RNA at specific time points, and sequenced their transcriptomes. First, molecular markers were used to locate and identify a 250 kb deletion on chromosome 10 in mutant crg (which carries a deletion at the Crg locus). Next, we identified differential expression of several disease resistance genes between Mock Inoculated (MI) and Inoculated (I) samples of "Sierra" leaf RNA within the 250 kb delineated region. Both marker assisted molecular profiling and RNA-seq were used to identify possible transcriptomic locations of interest regarding the resistance in the common bean to race 53. Identification of differential expression among samples in disease resistance clusters in the bean genome may elucidate significant genes underlying rust resistance. Along with preserving favorable traits in the crop, the current research may also aid in global sustainability of food stocks necessary for many populations.

Complete Genome Sequence of Spiroplasma citri Strain R8-A2T, Causal Agent of Stubborn Disease in Citrus Species.

Spiroplasma citri causes stubborn disease in Citrus spp. and diseases in other plants. Here, we report the nucleotide sequence of the 1,599,709-bp circular chromosome and two plasmids of S. citri strain R8-A2T This information will facilitate analyses to understand spiroplasmal pathogenicity and evolutionary adaptations to lifestyles in plants and arthropod hosts.

Optimization of the HyPer sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H2 O2 ) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H2 O2 dynamics in real time during important stages of the plant infection process.

Generic names in Magnaporthales.

The order Magnaporthales comprises about 200 species and includes the economically and scientifically important rice blast fungus and the take-all pathogen of cereals, as well as saprotrophs and endophytes. Recent advances in phylogenetic analyses of these fungi resulted in taxonomic revisions. In this paper we list the 28 currently accepted genera in Magnaporthales with their type species and available gene and genome resources. The polyphyletic Magnaporthe 1972 is proposed for suppression, and Pyricularia 1880 and Nakataea 1939 are recommended for protection as the generic names for the rice blast fungus and the rice stem rot fungus, respectively. The rationale for the recommended names is also provided. These recommendations are made by the Pyricularia/Magnaporthe Working Group established under the auspices of the International Commission on the Taxonomy of Fungi (ICTF).

Crucial Roles of Abscisic Acid Biogenesis in Virulence of Rice Blast Fungus Magnaporthe oryzae.

Rice suffers dramatic yield losses due to blast pathogen Magnaporthe oryzae. Pseudomonas chlororaphis EA105, a bacterium that was isolated from the rice rhizosphere, inhibits M. oryzae. It was shown previously that pre-treatment of rice with EA105 reduced the size of blast lesions through jasmonic acid (JA)- and ethylene (ETH)-mediated ISR. Abscisic acid (ABA) acts antagonistically toward salicylic acid (SA), JA, and ETH signaling, to impede plant defense responses. EA105 may be reducing the virulence of M. oryzae by preventing the pathogen from up-regulating the key ABA biosynthetic gene NCED3 in rice roots, as well as a ß-glucosidase likely involved in activating conjugated inactive forms of ABA. However, changes in total ABA concentrations were not apparent, provoking the question of whether ABA concentration is an indicator of ABA signaling and response. In the rice-M. oryzae interaction, ABA plays a dual role in disease severity by increasing plant susceptibility and accelerating pathogenesis in the fungus itself. ABA is biosynthesized by M. oryzae. Further, exogenous ABA increased spore germination and appressoria formation, distinct from other plant growth regulators. EA105, which inhibits appressoria formation, counteracted the virulence-promoting effects of ABA on M. oryzae. The role of endogenous fungal ABA in blast disease was confirmed through the inability of a knockout mutant impaired in ABA biosynthesis to form lesions on rice. Therefore, it appears that EA105 is invoking multiple strategies in its protection of rice from blast including direct mechanisms as well as those mediated through plant signaling. ABA is a molecule that is likely implicated in both tactics.

Complete Genome Sequence of Spiroplasma turonicum Strain Tab4cT, a Parasite of a Horse Fly, Haematopota sp. (Diptera: Tabanidae).

Spiroplasma turonicum was isolated from a Haematopota sp. fly in France. We report the nucleotide sequence of the circular chromosome of strain Tab4c(T). The genome information will facilitate evolutionary studies of spiroplasmas, including symbionts of insects and ticks and pathogens of plants, insects, crustaceans, and humans.

Complete Genome Sequence of Spiroplasma kunkelii Strain CR2-3x, Causal Agent of Corn Stunt Disease in Zea mays L.

Spiroplasma kunkelii causes corn stunt disease of Zea mays L. in the Americas. Here, we report the nucleotide sequence of the 1,463,926-bp circular chromosome and four plasmids of strain CR2-3x. This information will facilitate studies of Spiroplasma pathogenicity and evolutionary adaptations to transkingdom parasitism in plants and insect vectors.

Plant biomass degradation by fungi.

Plant biomass degradation by fungi has implications for several fields of science. The enzyme systems employed by fungi for this are broadly used in various industrial sectors such as food & feed, pulp & paper, detergents, textile, wine, and more recently biofuels and biochemicals. In addition, the topic is highly relevant in the field of plant pathogenic fungi as they degrade plant biomass to either gain access to the plant or as carbon source, resulting in significant crop losses. Finally, fungi are the main degraders of plant biomass in nature and as such have an essential role in the global carbon cycle and ecology in general. In this review we provide a global view on the development of this research topic in saprobic ascomycetes and basidiomycetes and in plant pathogenic fungi and link this to the other papers of this special issue on plant biomass degradation by fungi.

Natural rice rhizospheric microbes suppress rice blast infections.

BACKGROUND: The natural interactions between plant roots and their rhizospheric microbiome are vital to plant fitness, modulating both growth promotion and disease suppression. In rice (Oryza sativa), a globally important food crop, as much as 30% of yields are lost due to blast disease caused by fungal pathogen Magnaporthe oryzae. Capitalizing on the abilities of naturally occurring rice soil bacteria to reduce M. oryzae infections could provide a sustainable solution to reduce the amount of crops lost to blast disease. RESULTS: Naturally occurring root-associated rhizospheric bacteria were isolated from California field grown rice plants (M-104), eleven of which were taxonomically identified by 16S rRNA gene sequencing and fatty acid methyl ester (FAME) analysis. Bacterial isolates were tested for biocontrol activity against the devastating foliar rice fungal pathogen, M. oryzae pathovar 70-15. In vitro, a Pseudomonas isolate, EA105, displayed antibiosis through reducing appressoria formation by nearly 90% as well as directly inhibiting fungal growth by 76%. Although hydrogen cyanide (HCN) is a volatile commonly produced by biocontrol pseudomonads, the activity of EA105 seems to be independent of its HCN production. During in planta experiments, EA105 reduced the number of blast lesions formed by 33% and Pantoea agglomerans isolate, EA106 by 46%. Our data also show both EA105 and EA106 trigger jasmonic acid (JA) and ethylene (ET) dependent induced systemic resistance (ISR) response in rice. CONCLUSIONS: Out of 11 bacteria isolated from rice soil, pseudomonad EA105 most effectively inhibited the growth and appressoria formation of M. oryzae through a mechanism that is independent of cyanide production. In addition to direct antagonism, EA105 also appears to trigger ISR in rice plants through a mechanism that is dependent on JA and ET signaling, ultimately resulting in fewer blast lesions. The application of native bacteria as biocontrol agents in combination with current disease protection strategies could aid in global food security.