Experimental host and vector ranges of the emerging maize yellow mosaic polerovirus.

Maize yellow mosaic virus (MaYMV) is an emerging polerovirus that has been detected in maize, other cereal crops and weedy grass species in Asia, Africa, and the Americas. Disease symptoms in maize include prominent leaf tip reddening and stunting. Infection by MaYMV has been reported to reduce plant growth and yields by 10-30% in some instances. In this study, an experimental host range for MaYMV among agronomically important cereal crops and common grasses was established. Additional aphid species were assessed as potential vectors for MaYMV and their transmission efficiencies were determined. Here we report oats, foxtail millet, barley, and rye as new experimental cereal crop hosts of MaYMV in addition to confirming the previously reported hosts of corn, sorghum, wheat, and broom millet. Four of the nine other grass species evaluated were also identified as suitable experimental hosts for MaYMV: ryegrass, switchgrass, green foxtail, and sand love grass. Interestingly, no visible symptoms were present in any of the infected hosts besides the susceptible maize control. Vector range studies identified the greenbug aphid, Schizaphis graminum, as a new vector of MaYMV, though transmission efficiency was lower than the previously reported Rhopalosiphum maidis vector and similar to the other known aphid vector, R. padi. Given MaYMV's global ubiquity, ability to evade detection, and broad host range, further characterization of yield impacts and identification of viable control strategies are desirable.

Affinity Purification-Mass Spectrometry Identifies a Novel Interaction between a Polerovirus and a Conserved Innate Immunity Aphid Protein that Regulates Transmission Efficiency.

The vast majority of plant viruses are transmitted by insect vectors, with many crucial aspects of the transmission process being mediated by key protein-protein interactions. Still, very few vector proteins interacting with viruses have been identified and functionally characterized. Potato leafroll virus (PLRV) is transmitted most efficiently by Myzus persicae, the green peach aphid, in a circulative, non-propagative manner. Using affinity purification coupled to high-resolution mass spectrometry (AP-MS), we identified 11 proteins from M. persicaedisplaying a high probability of interaction with PLRV and an additional 23 vector proteins with medium confidence interaction scores. Three of these aphid proteins were confirmed to directly interact with the structural proteins of PLRV and other luteovirid species via yeast two-hybrid. Immunolocalization of one of these direct PLRV-interacting proteins, an orthologue of the human innate immunity protein complement component 1 Q subcomponent-binding protein (C1QBP), shows that MpC1QBP partially co-localizes with PLRV in cytoplasmic puncta and along the periphery of aphid gut epithelial cells. Artificial diet delivery to aphids of a chemical inhibitor of C1QBP leads to increased PLRV acquisition by aphids and subsequently increased titer in inoculated plants, supporting a role for C1QBP in the acquisition and transmission efficiency of PLRV by M. persicae. This study presents the first use of AP-MS for the in vivo isolation of a functionally relevant insect vector-virus protein complex. MS data are available from ProteomeXchange.org using the project identifier PXD022167.

Looking Through the Lens of 'Omics Technologies: Insights Into the Transmission of Insect Vector-borne Plant Viruses.

Insects in the orders Hemiptera and Thysanoptera transmit viruses and other pathogens associated with the most serious diseases of plants. Plant viruses transmitted by these insects target similar tissues, genes, and proteins within the insect to facilitate plant-to-plant transmission with some degree of specificity at the molecular level. 'Omics experiments are becoming increasingly important and practical for vector biologists to use towards better understanding the molecular mechanisms and biochemistry underlying transmission of these insect-borne diseases. These discoveries are being used to develop novel means to obstruct virus transmission into and between plants. In this chapter, we summarize 'omics technologies commonly applied in vector biology and the important discoveries that have been made using these methods, including virus and insect proteins involved in transmission, as well as the tri-trophic interactions involved in host and vector manipulation. Finally, we critically examine the limitations and new horizons in this area of research, including the role of endosymbionts and insect viruses in virus-vector interactions, and the development of novel control strategies.

Preservation of Monilinia fructicola Genotype Diversity Within Fungal Cankers.

Monilinia fructicola is a destructive pathogen causing brown rot on stone fruits worldwide. Though it is best known as a fruit rot pathogen, M. fructicola also causes blossom blight and, subsequently, twig cankers in the spring. Orchard management strategies often overlook cankers as an inoculum source, though they are an inoculum source of both blossom and fruit infections. In this study, we analyzed the role of cankers as storage structures for diverse genotypes of M. fructicola, examining whether multiple genotypes can be transmitted from blossom to canker. Fungal spores from blossoms, and 2 months later from their corresponding cankers, were collected from a conventional and an unsprayed orchard in 2015 and 2016. Simple sequence repeat markers were used to genotype 10 to 20 single spores from each of four blossom/canker pairs per orchard. Individual blossoms and cankers were detected containing up to four and five genotypes, respectively. The average number of genotypes in blossoms and corresponding cankers were not significantly different (P = 0.690) across both years and farms, showing that a bottleneck for genetic diversity was not generated during the transition from blossom to canker. The average number of genotypes unique to blossom or canker was not significantly different (P = 0.569) and no significant effect of farm (P = 0.961) or year (P = 0.520) was observed, although blossoms had a numerically greater number of unique genotypes in both cases. In conclusion, a single blossom may be infected by one or more genotypes of M. fructicola, and this diversity is being preserved in the corresponding canker. This information implicates M. fructicola cankers as diversity storehouses, and may also apply to other Monilinia spp. and fungal diseases that initiate in reproductive tissue.

Effect of Fungicide Applications on Monilinia fructicola Population Diversity and Transposon Movement.

In this study, we investigated whether fungicide-induced mutagenesis previously reported in Monilinia fructicola could accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied to nectarine trees at weekly intervals for approximately 3 months between bloom and harvest in both 2013 and 2014. Fungicides were applied at half-label rate to allow recovery of isolates and to increase chances of sublethal dose exposure. One block was left unsprayed as a control. In total, 608 single-spore isolates were obtained from blighted blossoms, cankers, and fruit to investigate phenotypic (fungicide resistance) and genotypic (simple-sequence repeat [SSR] loci and gene region) changes. In both years, populations from fungicide-treated and untreated fruit were not statistically different in haploid gene diversity (P = 0.775 for 2013 and P = 0.938 for 2014), allele number (P = 0.876 for 2013 and P = 0.406 for 2014), and effective allele number (P = 0.861 for 2013 and P = 0.814 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments revealed no changes in SSR analysis or evidence for induced Mftc1 transposon translocation. No indirect evidence for increased genetic diversity in the form of emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil was detected. High levels of population diversity in all treatments provided evidence for sexual recombination of this pathogen in the field, despite apparent absence of apothecia in the orchard. Our results indicate that fungicide-induced, genetic changes may not occur or not occur as readily in field populations as they do under continuous exposure to sublethal doses in vitro.

Characterization of Botrytis cinerea Isolates from Strawberry with Reduced Sensitivity to Polyoxin D Zinc Salt.

Polyoxin D is a Fungicide Resistance Action Committee (FRAC) code 19 fungicide that was recently registered for gray mold control of strawberry in the United States. In this study, we determined the sensitivity to polyoxin D zinc salt (hereafter, polyoxin D) of Botrytis cinerea isolates from 41 commercial strawberry farms in South Carolina, North Carolina, Maryland, Virginia, and Ohio and investigated the fitness of sensitive (S) and reduced sensitive (RS) isolates. Relative mycelial growth ranged between 0 and over 100% on malt extract agar amended with a discriminatory dose of polyoxin D at 5 µg/ml. Isolates that grew more than 70% at that dose were designated RS and were found in three of the five states. The 50% effective dose (EC50) values of three S and three RS isolates ranged from 0.59 to 2.27 and 4.6 to 5.8 µg/ml, respectively. The three RS isolates grew faster on detached tomato fruit treated with Ph-D WDG at recommended label dosage than S isolates (P < 0.008). In all, 25 randomly selected RS isolates exhibited reduced sporulation ability (P < 0.0001) and growth rate (P < 0.0001) but increased production of sclerotia (P < 0.0386) compared with 25 S isolates. Of 10 isolates tested per phenotype, the number of RS isolates producing sporulating lesions on apple, tomato, and strawberry was significantly lower compared with S isolates (P < 0.0001 for each fruit type). The results of this study indicate that resistance management is necessary for fungicides containing polyoxin D. To our knowledge, this is the first study demonstrating reduced sensitivity to FRAC 19 fungicides in B. cinerea isolates from the United States.

Soybean Aphid (Hemiptera: Aphididae) Feeding Behavior is Largely Unchanged by Soybean Mosaic Virus but Significantly Altered by the Beetle-Transmitted Bean Pod Mottle Virus.

The soybean aphid (Aphis glycines Matsumura) is an economically important invasive pest of soybean. In addition to damage caused by soybean aphid feeding on the phloem sap, this insect also transmits many plant viruses, including soybean mosaic virus (SMV). Previous work has shown that plant viruses can change plant host phenotypes to alter the behavior of their insect vectors to promote virus spread, known as the vector manipulation hypothesis. In this study, we used electropenetography (EPG) to examine the effects of two plant viruses on soybean aphid feeding behavior: SMV, which is transmitted by many aphid species including the soybean aphid, and bean pod mottle virus (BPMV), which is transmitted by chrysomelid and some coccinellid beetles but not aphids. These two viruses often co-occur in soybean production and can act synergistically. Surprisingly, our results showed little to no effect of SMV on soybean aphid feeding behaviors measured by EPG, but profound differences were observed in aphids feeding on BPMV-infected plants. Aphids took longer to find the vascular bundle of BPMV-infected plants, and once found, spent more time entering and conditioning the phloem than ingesting phloem sap. Interestingly, these observed alterations are similar to those of aphids feeding on insect-resistant soybean plants. The cause of these changes in feeding behavior is not known, and how they impact virus transmission and soybean aphid populations in the field will require further study.

The tomato yellow leaf curl virus C4 protein alters the expression of plant developmental genes correlating to leaf upward cupping phenotype in tomato.

Tomato yellow leaf curl virus (TYLCV), a monopartite begomovirus in the family Geminiviridae, is efficiently transmitted by the whitefly, Bemisia tabaci, and causes serious economic losses to tomato crops around the world. TYLCV-infected tomato plants develop distinctive symptoms of yellowing and leaf upward cupping. In recent years, excellent progress has been made in the characterization of TYLCV C4 protein function as a pathogenicity determinant in experimental plants, including Nicotiana benthamiana and Arabidopsis thaliana. However, the molecular mechanism leading to disease symptom development in the natural host plant, tomato, has yet to be characterized. The aim of the current study was to generate transgenic tomato plants expressing the TYLCV C4 gene and evaluate differential gene expression through comparative transcriptome analysis between the transgenic C4 plants and the transgenic green fluorescent protein (Gfp) gene control plants. Transgenic tomato plants expressing TYLCV C4 developed phenotypes, including leaf upward cupping and yellowing, that are similar to the disease symptoms expressed on tomato plants infected with TYLCV. In a total of 241 differentially expressed genes identified in the transcriptome analysis, a series of plant development-related genes, including transcription factors, glutaredoxins, protein kinases, R-genes and microRNA target genes, were significantly altered. These results provide further evidence to support the important function of the C4 protein in begomovirus pathogenicity. These transgenic tomato plants could serve as basic genetic materials for further characterization of plant receptors that are interacting with the TYLCV C4.

Polerovirus N-terminal readthrough domain structures reveal molecular strategies for mitigating virus transmission by aphids.

Poleroviruses, enamoviruses, and luteoviruses are icosahedral, positive sense RNA viruses that cause economically important diseases in food and fiber crops. They are transmitted by phloem-feeding aphids in a circulative manner that involves the movement across and within insect tissues. The N-terminal portion of the viral readthrough domain (NRTD) has been implicated as a key determinant of aphid transmission in each of these genera. Here, we report crystal structures of the NRTDs from the poleroviruses turnip yellow virus (TuYV) and potato leafroll virus (PLRV) at 1.53-Å and 2.22-Å resolution, respectively. These adopt a two-domain arrangement with a unique interdigitated topology and form highly conserved dimers that are stabilized by a C-terminal peptide that is critical for proper folding. We demonstrate that the PLRV NRTD can act as an inhibitor of virus transmission and identify NRTD mutant variants that are lethal to aphids. Sequence conservation argues that enamovirus and luteovirus NRTDs will follow the same structural blueprint, which affords a biological approach to block the spread of these agricultural pathogens in a generalizable manner.