Melvin D. Bolton

Tobacco leaf spot and root rot caused by Rhizoctonia solani Kühn.

UNLABELLEDnRhizoctonia solani Kühn is a soil-borne fungal pathogen that causes disease in a wide range of plants worldwide. Strains of the fungus are traditionally grouped into genetically isolated anastomosis groups (AGs) based on hyphal anastomosis reactions. This article summarizes aspects related to the infection process, colonization of the host and molecular mechanisms employed by tobacco plants in resistance against R. solani diseases.nnnTAXONOMYnTeleomorph: Thanatephorus cucumeris (Frank) Donk; anamorph: Rhizoctonia solani Kühn; Kingdom Fungi; Phylum Basidiomycota; Class Agaricomycetes; Order Cantharellales; Family Ceratobasidiaceae; genus Thanatephorus.nnnIDENTIFICATIONnSomatic hyphae in culture and hyphae colonizing a substrate or host are first hyaline, then buff to dark brown in colour when aging. Hyphae tend to form at right angles at branching points that are usually constricted. Cells lack clamp connections, but possess a complex dolipore septum with continuous parenthesomes and are multinucleate. Hyphae are variable in size, ranging from 3 to 17 µm in diameter. Although the fungus does not produce any conidial structure, ellipsoid to globose, barrel-shaped cells, named monilioid cells, 10-20 µm wide, can be produced in chains and can give rise to sclerotia. Sclerotia are irregularly shaped, up to 8-10 mm in diameter and light to dark brown in colour.nnnDISEASE SYMPTOMSnSymptoms in tobacco depend on AG as well as on the tissue being colonized. Rhizoctonia solani AG-2-2 and AG-3 infect tobacco seedlings and cause damping off and stem rot. Rhizoctonia solani AG-3 causes sore shin and target spot in mature tobacco plants. In general, water-soaked lesions start on leaves and extend up the stem. Stem lesions vary in colour from brown to black. During late stages, diseased leaves are easily separated from the plant because of severe wilting. In seed beds, disease areas are typically in the form of circular to irregular patches of poorly growing, yellowish and/or stunted seedlings.nnnRESISTANCEnKnowledge is scarce regarding the mechanisms associated with resistance to R. solani in tobacco. However, recent evidence suggests a complex response that involves several constitutive factors, as well as induced barriers controlled by multiple defence pathways.nnnMANAGEMENTnThis fungus can survive for many years in soil as mycelium, and also by producing sclerotia, which makes the management of the disease using conventional means very difficult. Integrated pest management has been most successful; it includes timely fungicide applications, crop rotation and attention to soil moisture levels. Recent developments in biocontrol may provide other tools to control R. solani in tobacco.

Generation and Characterization of a Sugarbeet Transcriptome and Transcript-Based SSR Markers

Sugarbeet is a major source of refined sucrose and increasingly grown for biofuel production. Demand for higher productivity for this crop requires greater knowledge of sugarbeet physiology, pathology, and genetics, which can be advanced by the development of new genomic resources. Towards this end, a sugarbeet transcriptome of expressed genes from leaf and root tissues at varying stages of development and production, and after elicitation with jasmonic acid (JA) or salicylic acid (SA), was constructed and used to generate simple sequence repeat (SSR) markers. The transcriptome was generated via paired‐end RNA sequencing and contains 82,404 unigenes. A total of 37,207 unigenes were annotated, of which 9480 were functionally classified using clusters of orthologous groups (COG) annotations, 17,191 were classified into biological process, molecular function, or cellular component using gene ontology (GO) terms, and 17,409 were assigned to 126 metabolic pathways using Kyoto Encyclopedia of Genes and Genomes (KEGG) identifiers. A SSR search of the transcriptome identified 7680 SSRs, including 6577 perfect SSRs, of which 3834 were located in unigenes with ungapped sequence. Primer‐pairs were designed for 288 SSR loci, and 72 of these primer‐pairs were tested for their ability to detect polymorphisms. Forty‐three primer‐pairs detected single polymorphic loci and effectively distinguished diversity among eight B. vulgaris genotypes. The transcriptome and SSR markers provide additional, public domain genomic resources for an important crop plant and can be used to increase understanding of the functional elements of the sugarbeet genome, aid in discovery of novel genes, facilitate RNA‐sequencing based expression research, and provide new tools for sugarbeet genetic research and selective breeding.

Construction of Hairpin RNA-Expressing Vectors for RNA-Mediated Gene Silencing in Fungi

RNA-mediated gene silencing is one of the major tools for functional genomics in fungi and can be achieved by transformation with constructs that express hairpin (hp) RNA with sequences homologous to the target gene(s). To make an hpRNA expression construct, a portion of the target gene can be amplified by PCR and cloned into a vector as an inverted repeat. The generic gene-silencing vectors such as the pSilent1 and pSGate1 have been developed and are available for RNA-mediated gene-silencing studies. In this protocol, we describe construction of hpRNA-expressing constructs using both pSilent1 and pSGate1. With pSilent1, the PCR products of the target gene are inserted into the vector by conventional cloning (i.e., restriction enzyme digestion and ligation). For pSGate1, the PCR products of the target gene are inserted into the vector through the Gateway-directed recombination system. In this chapter, we describe the construction of RNAi vectors for RNA-mediated gene silencing using both pSilent1 and pSGate1.

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 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 Elsinoё 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. Originality and significance statement Genes involved in secondary metabolite (SM) production are often clustered together to form biosynthetic pathways. These pathways frequently have highly conserved keystone enzymes which can complicate allocation of a biosynthetic gene cluster (BGC) to the cognate SM. In our study, we utilized a combination of comparative genomics, phylogenetic analyses and biochemical approaches to reliably identify BGCs for perylenequinone toxins and DHN-melanin in multiple plant pathogenic fungi. Furthermore, we show that earlier studies that aimed to identify these perylenequinone pathways were misdirected and actually reported DHN-melanin biosynthetic pathways. Our study outlines a reliable approach to successfully identify fungal SM pathways.

Short- and long-term changes in sugarbeet (Beta vulgaris L.) gene expression due to postharvest jasmonic acid treatment - Data

Jasmonic acid is a natural plant hormone that induces native defense responses in plants. Sugarbeet (Beta vulgaris L.) root unigenes that were differentially expressed 2 and 60 days after a postharvest jasmonic acid treatment are presented. Data include changes in unigene expression relative to water-treated controls, unigene annotations against nonredundant (Nr), Swiss-Prot, Clusters of Orthologous Groups (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases, and unigene annotations with Gene Ontology (GO) terms. Putative defense unigenes are compiled and annotated against the sugarbeet genome. Differential gene expression data were generated by RNA sequencing. Interpretation of the data is available in the research article, “Jasmonic acid causes short- and long-term alterations to the transcriptome and the expression of defense genes in sugarbeet roots” (K.K. Fugate, L.S. Oliveira, J.P. Ferrareze, M.D. Bolton, E.L. Deckard, F.L. Finger, 2017) [1]. Public dissemination of this dataset will allow further analyses of the data.

High-throughput SuperSAGE for gene expression analysis of Nicotiana tabacum – Rhizoctonia solani interaction

ObjectiveThe ubiquitous soil pathogen Rhizoctonia solani causes serious diseases in different plant species. Despite the importance of this disease, little is known regarding the molecular basis of susceptibility. SuperSAGE technology and next-generation sequencing were used to generate transcript libraries during the compatible Nicotiana tabacum–R. solani interaction. Also, we used the post-transcriptional silencing to evaluate the function of a group of important genes.ResultsA total of 8960 and 8221 unique Tag sequences identified as differentially up- and down-regulated were obtained. Based on gene ontology classification, several annotated UniTags corresponded to defense response, metabolism and signal transduction. Analysis of the N. tabacum transcriptome during infection identified regulatory genes implicated in a number of hormone pathways. Silencing of an mRNA induced by salicylic acid reduced the susceptibility of N. tabacum to R. solani. We provide evidence that the salicylic acid pathway was involved in disease development. This is important for further development of disease management strategies caused by this pathogen.

USE OF JASMONIC ACID AND SALICYLIC ACID TO INHIBIT GROWTH OF SUGARBEET STORAGE ROT PATHOGENS

Jasmonic acid (JA) and salicylic acid (SA) are endogenous plant hormones that induce native plant defense responses and provide protection against a wide range of diseases. Previously, JA, applied after harvest, was shown to protect sugarbeet roots against the storage pathogens, Botrytis cinerea, Penicillium claviforme, and Phoma betae by reducing the severity of rot symptoms due to these pathogens by 51, 44, and 71%, respectively (Fugate et al., 2012, Postharvest Biol. Technol., 65:1-4). Research was conducted to determine the ability of SA to protect sugarbeet roots from these storage rot pathogens and to investigate the use of preharvest treatments of JA or methyl jasmonate (MeJA), a low cost derivative of JA, to reduce storage rot due to B. cinerea, P. claviforme, or P. betae. The effect of water stress on severity of rot symptoms due to B. cinerea, P. claviforme, and P. betae was also investigated. SA, applied after harvest at concentrations of 0.01, 0.1, 1.0 or 10 mM, had no effect on the severity of storage rot symptoms in roots obtained from healthy, unstressed plants after inoculation with B. cinerea, P. claviforme, and P. betae. However, when roots were obtained from water-stressed plants, 0.01 to 10 mM SA reduced the severity of rot symptoms due to B. cinerea by 49—58%, P. claviforme by 30—53%, and P. betae by 47—74%. All concentrations of SA provided statistically similar reductions in the weight of rotted tissue for each of the three pathogens, and on average, postharvest SA treatment reduced the weight of rotted tissue due to B. cinerea, P. claviforme, and P. betae by 54, 45, and 58% respectively. SA reduced the weight of rotted tissue in roots from water-stressed plants by reducing lesion size, but had no effect on the incidence of infection. The ability of SA to reduce rot severity in water-stressed roots but not in roots harvested from plants that received sufficient water prior to harvest suggests that SA mitigated the negative effects of water stress, but did not directly protect roots against storage pathogens. Results from SA experiments described above suggested that drought stress increased root rot severity due to B. cinerea, P. claviforme, and P. betae. To verify this, water was withheld from greenhouse-grown plants, roots were harvested two days after plants were severely wilted, and the harvested roots were inoculated with B. cinerea, P. claviforme, or P. betae. Relative to roots obtained from well-watered plants, roots from water-stressed plants had 2.3-fold more rot due to B. cinerea, 1.4-fold more rot due to P. claviforme, and 2.4-fold more rot due to P. betae. Field-grown roots from water-stressed plants also exhibited increases in rot severity due to B. cinerea and P. betae, but not P. claviforme. The ability of preharvest JA treatments to reduce storage rot was determined by application of 0.01 or 10 μM JA or MeJA to foliage 3, 7, 14, or 30 days prior to harvest. Although this research is not complete, preliminary results suggest that preharvest JA or MeJA treatments reduce the severity of storage rot due to B. cinerea, P. claviforme, and P. betae. In general, 0.01 μM JA was more effective in reducing storage rot than 10 μM JA. As was observed previously for postharvest JA treatments, preharvest JA treatments were most effective against P. betae and least effective against P. claviforme.