Interactions between \u3cem\u3eAlfalfa mosaic virus\u3c/em\u3e and \u3cem\u3eSoybean mosaic virus\u3c/em\u3e in soybean

Viral synergism occurs when two or more unrelated viruses simultaneously infect the same plant and the multiplication of one of the viruses is enhanced. This is generally associated with no change(s) in multiplication of the other viruses involved. Synergism also results in intensification of symptoms. In mixed-infection, viruses may also interact in an antagonistic manner, where one virus suppresses the replication or accumulation of another virus. This phenomenon is uncommon, and only two cases have been reported where the coat protein (CP) accumulation of one of the viruses has decreased. A number of synergistic interactions studied involve viruses belonging to the Potyviridae family. The increase in CP accumulation of the non-potyviruses in such an interaction has been attributed to the effect of the helper-component proteinase (HC-Pro) of potyviruses. Plant antiviral defense mechanism called “gene silencing”. HC-Pro is known as a strong suppressor of gene silencing and represents the first identified and characterized plant viral suppressor of gene silencing. The ability of Soybean mosaic virus (SMV), a member of the Potyviridae family, to interact synergistically with Bean pod mottle virus (BPMV) and Cowpea mosaic virus (CPMV) in mixed-infection in soybean has been demonstrated, but no change in the level of accumulation of CP of SMV was reported. In addition to SMV, soybean is infected by many other potyviruses or non-potyviruses, including Alfalfa mosaic virus (AMV). This research was aimed at studying the interaction of SMV with AMV in mixed-infection in soybean. Two biologically distinct SMV strains and three AMV isolates were used in this study and their interactions in mixed-infection in two different cultivars of soybeans (Williams 82 and Lee 68) were investigated. It was demonstrated that (a) mixed-infection between AMV and SMV can be easily established, irrespective of sequential or simultaneous inoculation of the two viruses; (b) based on CP accumulation and disease phenotype, AMV interaction with SMV is synergistic resulting in enhancement in symptom severity and AMV CP accumulation; (c) synergistic interaction of AMV with SMV is strain and cultivar independent; (d) interaction of SMV with AMV is antagonistic, which is also strain and soybean cultivar-independent

Genome analysis of the ubiquitous boxwood pathogen Pseudonectria foliicola

Boxwood (Buxus spp.) are broad-leaved, evergreen landscape plants valued for their longevity and ornamental qualities. Volutella leaf and stem blight, caused by the ascomycete fungi Pseudonectria foliicola and P. buxi, is one of the major diseases affecting the health and ornamental qualities of boxwood. Although this disease is less severe than boxwood blight caused by Calonectria pseudonaviculata and C. henricotiae, its widespread occurrence and disfiguring symptoms have caused substantial economic losses to the ornamental industry. In this study, we sequenced the genome of P. foliicola isolate ATCC13545 using Illumina technology and compared it to other publicly available fungal pathogen genomes to better understand the biology of this organism. A de novo assembly estimated the genome size of P. foliicola at 28.7 Mb (425 contigs; N50 = 184,987 bp; avg. coverage 188×), with just 9,272 protein-coding genes. To our knowledge, P. foliicola has the smallest known genome within the Nectriaceae. Consistent with the small size of the genome, the secretome, CAzyme and secondary metabolite profiles of this fungus are reduced relative to two other surveyed Nectriaceae fungal genomes: Dactylonectria macrodidyma JAC15-245 and Fusarium graminearum Ph-1. Interestingly, a large cohort of genes associated with reduced virulence and loss of pathogenicity was identified from the P. foliicola dataset. These data are consistent with the latest observations by plant pathologists that P. buxi and most likely P. foliicola, are opportunistic, latent pathogens that prey upon weak and stressed boxwood plants

Development of a Real-Time Microchip PCR System for Portable Plant Disease Diagnosis

Rapid and accurate detection of plant pathogens in the field is crucial to prevent the proliferation of infected crops. Polymerase chain reaction (PCR) process is the most reliable and accepted method for plant pathogen diagnosis, however current conventional PCR machines are not portable and require additional post-processing steps to detect the amplified DNA (amplicon) of pathogens. Real-time PCR can directly quantify the amplicon during the DNA amplification without the need for post processing, thus more suitable for field operations, however still takes time and require large instruments that are costly and not portable. Microchip PCR systems have emerged in the past decade to miniaturize conventional PCR systems and to reduce operation time and cost. Real-time microchip PCR systems have also emerged, but unfortunately all reported portable real-time microchip PCR systems require various auxiliary instruments. Here we present a stand-alone real-time microchip PCR system composed of a PCR reaction chamber microchip with integrated thin-film heater, a compact fluorescence detector to detect amplified DNA, a microcontroller to control the entire thermocycling operation with data acquisition capability, and a battery. The entire system is 25 × 16 × 8 cm(3) in size and 843 g in weight. The disposable microchip requires only 8-µl sample volume and a single PCR run consumes 110 mAh of power. A DNA extraction protocol, notably without the use of liquid nitrogen, chemicals, and other large lab equipment, was developed for field operations. The developed real-time microchip PCR system and the DNA extraction protocol were used to successfully detect six different fungal and bacterial plant pathogens with 100% success rate to a detection limit of 5 ng/8 µl sample

Population Genomics Provide Insights into the Global Genetic Structure of \u3ci\u3eColletotrichum graminicola\u3c/i\u3e, the Causal Agent of Maize Anthracnose

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and wholegenome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure

Population Genomics Provide Insights into the Global Genetic Structure of Colletotrichum graminicola, the Causal Agent of Maize Anthracnose

Understanding the genetic diversity and mechanisms underlying genetic variation in pathogen populations is crucial to the development of effective control strategies. We investigated the genetic diversity and reproductive biology of Colletotrichum graminicola isolates which infect maize by sequencing the genomes of 108 isolates collected from 14 countries using restriction site-associated DNA sequencing (RAD-seq) and whole-genome sequencing (WGS). Clustering analyses based on single-nucleotide polymorphisms revealed three genetic groups delimited by continental origin, compatible with short-dispersal of the pathogen and geographic subdivision. Intra- and intercontinental migration was observed between Europe and South America, likely associated with the movement of contaminated germplasm. Low clonality, evidence of genetic recombination, and high phenotypic diversity were detected. We show evidence that, although it is rare (possibly due to losses of sexual reproduction- and meiosis-associated genes) C. graminicola can undergo sexual recombination. Our results support the hypotheses that intra- and intercontinental pathogen migration and genetic recombination have great impacts on the C. graminicola population structure. IMPORTANCE Plant pathogens cause significant reductions in yield and crop quality and cause enormous economic losses worldwide. Reducing these losses provides an obvious strategy to increase food production without further degrading natural ecosystems; however, this requires knowledge of the biology and evolution of the pathogens in agroecosystems. We employed a population genomics approach to investigate the genetic diversity and reproductive biology of the maize anthracnose pathogen (Colletotrichum graminicola) in 14 countries. We found that the populations are correlated with their geographical origin and that migration between countries is ongoing, possibly caused by the movement of infected plant material. This result has direct implications for disease management because migration can cause the movement of more virulent and/or fungicide-resistant genotypes. We conclude that genetic recombination is frequent (in contrast to the traditional view of C. graminicola being mainly asexual), which strongly impacts control measures and breeding programs aimed at controlling this disease.This research was supported by grants AGL2015-66362-R, RTI2018-093611-B-100, and PID2021-125349NB-100, funded by the Ministry of Science and Innovation (MCIN) of Spain AEI/10.13039/501100011033; and by grant SA165U13 funded by the Junta de Castilla y Léon. F.R. was supported by grant FJC2020-043351-I financed by MCIN/AEI /10.13039/501100011033 and by the European Union NextGenerationEU/PRTR. R.B. was supported by the postdoctoral program of USAL (Program II). F.B.C.-F. was supported by grant BES-2016-078373, funded by MCIN/AEI/10.13039/501100011033. S.B. was supported by a fellowship program from the regional government of Castilla y León. W.B. was supported by a productivity fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 307855/2019-8). Genome sequencing was funded by the UNC Microbiome Core, which is funded in part by the Center for Gastrointestinal Biology and Disease (CGIBD P30 DK034987) and the UNC Nutrition Obesity Research Center (NORC P30 DK056350). P.D.E. was partially supported by the USDA National Institute of Food and Federal Appropriations under Project PEN04660 and accession no. 1016474.Peer reviewe

The Role of Cys2-His2 Zinc Finger Transcription Factors in Polyol Metabolism, Asexual Development and Fumonisin Biosynthesis in Fusarium verticillioides

The ascomycete Fusarium verticillioides (Sacc.) Nirenberg (teleomorph: Gibberella moniliformis Wineland) causes stalk and ear rots on maize worldwide. In addition to the economic losses due to reduced yield, the fungus produces fumonisins on infected corn. One of the unanswered questions in mycotoxin research is how fungi perceive and respond to various extracellular stimuli and produce mycotoxins. To date, extensive research has been performed on important signaling pathways that regulate mycotoxin biosynthesis, but little is known about the downstream target genes, notably transcription factors (TFs). While the roles of TFs have shown to be critical in eukaryotic transcription regulation, only a few have been characterized in F. verticillioides. TFs with zinc fingers have been reported in all living organisms, and in fungal species, members of the Cys2-Hys2 (C2H2) zinc finger TF family are predicted to be involved in cell differentiation, carbon utilization, and development. Using the available genomic resources, I constructed a library of C2H2 TF deletion mutants, and identified SDA1, FvFLBC and CHT1 genes with a potential role in carbon utilization, development and fumonisin B1 (FB1) biosynthesis. The Δsda1 strain showed complete growth inhibition when using sorbitol as the sole carbon source and produced higher levels of FB1 when grown on corn kernels. In addition, the Δsda1 strain produced less number of conidia compared to the wild-type progenitor. Through gene complementation, I also demonstrated that F. verticillioides SDA1 and Trichoderma reesei ACE1 are functionally conserved. FvFLBC acts as a regulator of asexual development but not FB1 biosynthesis. I also discovered that the FvFlbC N-terminus is critical for conidia production. CHT1 is associated with asexual development, fumonisin biosynthesis and pigmentation. Characterization of key signal transduction pathways, and more importantly the function of SDA1, FvFLBC and CHT1, should facilitate the elucidation of the mechanisms and regulations of growth, development, and secondary metabolism in F. verticillioides. The outcome of this study may help us determine how to minimize F. verticillioides contamination of crops and the resulting mycotoxins, providing safer and higher value corn in the US and worldwide

Effects of tissue type and season on the detection of regulated sugarcane viruses by high throughput sequencing

Abstract High throughput sequencing (HTS) can supplement and may replace diagnostic tests for plant pathogens. However, the methodology and processing of HTS data must first be optimized and standardized to ensure the sensitivity and repeatability of the results. Importation of sugarcane into the United States is highly regulated, and sugarcane plants are subjected to strict quarantine measures and diagnostic testing, especially for the presence of certain viruses of regulatory concern. Here, we tested whether HTS could reliably detect four RNA and three DNA sugarcane viruses over three seasons (fall, winter, and spring) and in three tissue types (root, stem, and leaves). Using HTS on ribosomal depleted total RNA samples, we reliably detected RNA viruses in all tissue types and across all seasons, but we failed to confidently detect DNA viruses in some samples. We recommend that future optimization be employed to ensure the robust and reliable detection of all regulated sugarcane viruses by HTS

Sda1, a Cys₂-His₂ Zinc Finger Transcription Factor, Is Involved in Polyol Metabolism and Fumonisin B₁ Production in <i>Fusarium verticillioides</i>

<div><p>The ubiquitous ascomycete <i>Fusarium verticillioides</i> causes ear rot and stalk rot of maize, both of which reduce grain quality and yield. Additionally, <i>F. verticillioides</i> produces the mycotoxin fumonisin B₁ (FB₁) during infection of maize kernels, and thus potentially compromises human and animal health. The current knowledge is fragmentary regarding the regulation of FB₁ biosynthesis, particularly when considering interplay with environmental factors such as nutrient availability. In this study, <i>SDA1</i> of <i>F. verticillioides</i>, predicted to encode a Cys-2 His-2 zinc finger transcription factor, was shown to play a key role in catabolizing select carbon sources. Growth of the <i>SDA1</i> knock-out mutant (Δsda1) was completely inhibited when sorbitol was the sole carbon source and was severely impaired when exclusively provided mannitol or glycerol. Deletion of <i>SDA1</i> unexpectedly increased FB₁ biosynthesis, but reduced arabitol and mannitol biosynthesis, as compared to the wild-type progenitor. <i>Trichoderma reesei ACE1,</i> a regulator of cellulase and xylanase expression, complemented the <i>F. verticillioides</i> Δsda1 mutant, which indicates that Ace1 and Sda1 are functional orthologs. Taken together, the data indicate that Sda1 is a transcriptional regulator of carbon metabolism and toxin production in <i>F. verticillioides</i>.</p></div

Identification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus