Transcriptomic and metabolomic changes in postharvest sugarbeet roots reveal widespread metabolic changes in storage and identify genes potentially responsible for respiratory sucrose loss.

Endogenous metabolism is primarily responsible for losses in sucrose content and processing quality in postharvest sugarbeet roots. The genes responsible for this metabolism and the transcriptional changes that regulate it, however, are largely unknown. To identify genes and metabolic pathways that participate in postharvest sugarbeet root metabolism and the transcriptional changes that contribute to their regulation, transcriptomic and metabolomic profiles were generated for sugarbeet roots at harvest and after 12, 40 and 120 d storage at 5 and 12°C and gene expression and metabolite concentration changes related to storage duration or temperature were identified. During storage, 8656 genes, or 34% of all expressed genes, and 225 metabolites, equivalent to 59% of detected metabolites, were altered in expression or concentration, indicating extensive transcriptional and metabolic changes in stored roots. These genes and metabolites contributed to a wide range of cellular and molecular functions, with carbohydrate metabolism being the function to which the greatest number of genes and metabolites classified. Because respiration has a central role in postharvest metabolism and is largely responsible for sucrose loss in sugarbeet roots, genes and metabolites involved in and correlated to respiration were identified. Seventy-five genes participating in respiration were differentially expressed during storage, including two bidirectional sugar transporter SWEET17 genes that highly correlated with respiration rate. Weighted gene co-expression network analysis identified 1896 additional genes that positively correlated with respiration rate and predicted a pyruvate kinase gene to be a central regulator or biomarker for respiration rate. Overall, these results reveal the extensive and diverse physiological and metabolic changes that occur in stored sugarbeet roots and identify genes with potential roles as regulators or biomarkers for respiratory sucrose loss.

Histopathological Investigation of Varietal Responses to Cercospora beticola Infection Process on Sugar Beet Leaves.

Cercospora leaf spot (CLS) is the most destructive foliar disease in sugar beet (Beta vulgaris). It is caused by Cercospora beticola Sacc., a fungal pathogen that produces toxins and enzymes which affect membrane permeability and cause cell death during infection. In spite of its importance, little is known about the initial stages of leaf infection by C. beticola. Therefore, we investigated the progression of C. beticola on leaf tissues of susceptible and resistant sugar beet varieties at 12-h intervals during the first 5 days after inoculation using confocal microscopy. Inoculated leaf samples were collected and stored in DAB (3,3'-diaminobenzidine) solution until processed. Samples were stained with Alexa Fluor-488-WGA dye to visualize fungal structures. Fungal biomass accumulation, reactive oxygen species (ROS) production, and the area under the disease progress curve were evaluated and compared. ROS production was not detected on any variety before 36 h postinoculation (hpi). C. beticola biomass accumulation, percentage leaf cell death, and disease severity were all significantly greater in the susceptible variety compared with the resistant variety (P < 0.05). Conidia penetrated directly through stomata between 48 to 60 hpi and produced appressoria on stomatal guard cells at 60 to 72 hpi in susceptible and resistant varieties, respectively. Penetration of hyphae inside the parenchymatous tissues varied in accordance with time postinoculation and varietal genotypes. Overall, this study provides a detailed account to date of events leading to CLS disease development in two contrasting varieties.

Resistance to QoI and DMI Fungicides Does Not Reduce Virulence of C. beticola Isolates in North Central United States.

Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and is managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, Cercospora beticola, has been reported to be resistant to quinone outside inhibitors (QoIs) and to have reduced sensitive to demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. Isolates of C. beticola resistant to QoIs, DMIs, and both QoIs and DMIs were collected from fields in Foxhome, Minnesota, in 2017. Fitness of these resistant isolates was compared with that of QoI- and DMI-sensitive isolates in laboratory and greenhouse studies. In the lab, mycelial growth, spore production, and spore germination were measured. The results showed that resistant isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. In the greenhouse, six leaf-stage sugar beets were inoculated with a spore suspension made from each resistant group and incubated in separate humidity chambers. CLS disease severity was evaluated visually at 7, 14, and 21 days after inoculation (DAI), and the areas under disease progress curve (AUDPC) were calculated. Resistant isolates had significantly smaller AUDPC but still caused as high disease severity as the sensitive ones at 21 DAI. Although QoI- and/or DMI-resistant isolates had a relatively slower disease development, they still caused high disease severity and need to be factored in disease management practices.

Wounding rapidly alters transcription factor expression, hormonal signaling, and phenolic compound metabolism in harvested sugarbeet roots.

Injuries sustained by sugarbeet (Beta vulgaris L.) roots during harvest and postharvest operations seriously reduce the yield of white sugar produced from stored roots. Although wound healing is critically important to reduce losses, knowledge of these processes is limited for this crop as well as for roots in other species. To better understand the metabolic signals and changes that occur in wounded roots, dynamic changes in gene expression were determined by RNA sequencing and the activity of products from key genes identified in this analysis were determined in the 0.25 to 24 h following injury. Nearly five thousand differentially expressed genes that contribute to a wide range of cellular and molecular functions were identified in wounded roots. Highly upregulated genes included transcription factor genes, as well as genes involved in ethylene and jasmonic acid (JA) biosynthesis and signaling and phenolic compound biosynthesis and polymerization. Enzyme activities for key genes in ethylene and phenolic compound biosynthesis and polymerization also increased due to wounding. Results indicate that wounding causes a major reallocation of metabolism in sugarbeet taproots. Although both ethylene and JA are likely involved in triggering wound responses, the greater and more sustained upregulation of ethylene biosynthesis and signaling genes relative to those of JA, suggest a preeminence of ethylene signaling in wounded sugarbeet roots. Changes in gene expression and enzymes involved in phenolic compound metabolism additionally indicate that barriers synthesized to seal off wounds, such as suberin or lignin, are initiated within the first 24 h after injury.

Methyl jasmonate effects on sugarbeet root responses to postharvest dehydration.

BACKGROUND: Sugarbeet (Beta vulgaris L.) roots are stored under conditions that cause roots to dehydrate, which increases postharvest losses. Although exogenous jasmonate applications can reduce drought stress in intact plants, their ability to alleviate the effects of dehydration in postharvest sugarbeet roots or other stored plant products is unknown. Research was conducted to determine whether jasmonate treatment could mitigate physiological responses to dehydration in postharvest sugarbeet roots. METHODS: Freshly harvested sugarbeet roots were treated with 10 µM methyl jasmonate (MeJA) or water and stored under dehydrating and non-dehydrating storage conditions. Changes in fresh weight, respiration rate, wound healing, leaf regrowth, and proline metabolism of treated roots were investigated throughout eight weeks in storage. RESULTS: Dehydrating storage conditions increased root weight loss, respiration rate, and proline accumulation and prevented leaf regrowth from the root crown. Under dehydrating conditions, MeJA treatment reduced root respiration rate, but only in severely dehydrated roots. MeJA treatment also hastened wound-healing, but only in the late stages of barrier formation. MeJA treatment did not impact root weight loss or proline accumulation under dehydrating conditions or leaf regrowth under non-dehydrating conditions. Both dehydration and MeJA treatment affected expression of genes involved in proline metabolism. In dehydrated roots, proline dehydrogenase expression declined 340-fold, suggesting that dehydration-induced proline accumulation was governed by reducing proline degradation. MeJA treatment altered proline biosynthetic and catabolic gene expression, with greatest effect in non-dehydrated roots. Overall, MeJA treatment alleviated physiological manifestations of dehydration stress in stored roots, although the beneficial effects were small. Postharvest jasmonate applications, therefore, are unlikely to significantly reduce dehydration-related storage losses in sugarbeet roots.

Age-Dependent Resistance to Rhizoctonia solani in Sugar Beet.

Rhizoctonia crown and root rot of sugar beet (Beta vulgaris L.), caused by Rhizoctonia solani, continues to be one of the important concerns for the beet industry in Minnesota and North Dakota. Use of resistant cultivars is an important strategy in the management of R. solani in combination with seed treatment and timely fungicide application during the growing season. The objective of this greenhouse study was to determine how sugar beet plants responded to increasing age in resistance to R. solani. Each of three seed companies provided three commercial cultivars with varying R. solani resistance levels: susceptible, moderately resistant, and resistant. Seed were planted at a weekly interval to create different plant age groups from seed to 10-week-old plants, with growing degree days (GDD) ranging from 0 to 1,519 thermal time (°Cd). Seed and plants were all simultaneously inoculated with R. solani AG2-2-infested barley grains. Twenty-eight days after inoculation, plants were pulled and washed, and roots were evaluated for disease severity. All cultivars were highly susceptible to R. solani when inoculated at seed to 3 weeks old (0 to 464°Cd). At 4 and 5 weeks of plant age (617 to 766°Cd), resistant cultivars started to show significant resistance to R. solani. Proportion of the affected roots with disease score ≥ 5 followed a sigmoid response, declining with increased GDD in moderately resistant and resistant cultivars, whereas it continued to decline linearly with increased GDD in susceptible cultivars. This study demonstrated that sugar beet cultivars, regardless of their assigned level of R. solani resistance, were highly susceptible to the pathogen before they reached the six- to eight-leaf stage at 4 to 5 weeks (617 to 766°Cd) after planting. Therefore, additional protection in the form of seed treatment or fungicide application may be required to protect sensitive sugar beet seed and seedlings in fields with a history of R. solani under favorable environmental conditions.

Prevalence and Distribution of Beet Necrotic Yellow Vein Virus Strains in North Dakota and Minnesota.

Beet necrotic yellow vein virus (BNYVV) is the causal agent of rhizomania, a disease of global importance to the sugar beet industry. The most widely implemented resistance gene to rhizomania to date is Rz1, but resistance has been circumvented by resistance-breaking (RB) isolates worldwide. In an effort to gain greater understanding of the distribution of BNYVV and the nature of RB isolates in Minnesota and eastern North Dakota, sugar beet plants were grown in 594 soil samples obtained from production fields and subsequently were analyzed for the presence of BNYVV as well as coding variability in the viral P25 gene, the gene previously implicated in the RB pathotype. Baiting of virus from the soil with sugar beet varieties possessing no known resistance to rhizomania resulted in a disease incidence level of 10.6% in the region examined. Parallel baiting analysis of sugar beet genotypes possessing Rz1, the more recently introgressed Rz2, and with the combination of Rz1 + Rz2 resulted in a disease incidence level of 4.2, 1.0, and 0.8%, respectively. Virus sequences recovered from sugar beet bait plants possessing resistance genes Rz1 and/or Rz2 exhibited reduced genetic diversity in the P25 gene relative to those recovered from the susceptible genotype while confirming the hypervariable nature of the coding for amino acids (AAs) at position 67 and 68 in the P25 protein. In contrast to previous reports, we did not find an association between any one specific AA signature at these positions and the ability to circumvent Rz1-mediated resistance. The data document ongoing virulence development in BNYVV populations to previously resistant varieties and provide a baseline for the analysis of genetic change in the virus population that may accompany the implementation of new resistance genes to manage rhizomania.

Sensitivity of Rhizoctonia solani AG-2-2 from Sugar Beet to Fungicides.

Rhizoctonia damping-off and crown and root rot caused by Rhizoctonia solani are major diseases of sugar beet (Beta vulgaris L.) worldwide, and growers in the United States rely on fungicides for disease management. Sensitivity of R. solani to fungicides was evaluated in vitro using a mycelial radial growth assay and by evaluating disease severity on R. solani AG 2-2 inoculated plants treated with fungicides in the greenhouse. The mean concentration that caused 50% mycelial growth inhibition (EC50) values for baseline isolates (collected before the fungicides were registered for sugar beet) were 49.7, 97.1, 0.3, 0.2, and 0.9 µg ml-1 and for nonbaseline isolates (collected after registration and use of fungicides) were 296.1, 341.7, 0.9, 0.2, and 0.6 µg ml-1 for azoxystrobin, trifloxystrobin, pyraclostrobin, penthiopyrad, and prothioconazole, respectively. The mean EC50 values of azoxystrobin, trifloxystrobin, and pyraclostrobin significantly increased in the nonbaseline isolates compared with baseline isolates, with a resistant factor of 6.0, 3.5, and 3.0, respectively. Frequency of isolates with EC50 values >10 µg ml-1 for azoxystrobin and trifloxystrobin increased from 25% in baseline isolates to 80% in nonbaseline isolates. Although sensitivity of nonbaseline isolates of R. solani to quinone outside inhibitors decreased, these fungicides at labeled rates were still effective at controlling the pathogen under greenhouse conditions.

Characterization of Fusarium secorum, a new species causing Fusarium yellowing decline of sugar beet in north central USA.

This study characterized a novel sugar beet (Beta vulgaris L.) pathogen from the Red River Valley in north central USA, which was formally named Fusarium secorum. Molecular phylogenetic analyses of three loci (translation elongation factor1α, calmodulin, mitochondrial small subunit) and phenotypic data strongly supported the inclusion of F. secorum in the Fusarium fujikuroi species complex (FFSC). Phylogenetic analyses identified F. secorum as a sister taxon of F. acutatum and a member of the African subclade of the FFSC. Fusarium secorum produced circinate hyphae sometimes bearing microconidia and abundant corkscrew-shaped hyphae in culture. To assess mycotoxin production potential, 45 typical secondary metabolites were tested in F. secorum rice cultures, but only beauvericin was produced in detectable amounts by each isolate. Results of pathogenicity experiments revealed that F. secorum isolates are able to induce half- and full-leaf yellowing foliar symptoms and vascular necrosis in roots and petioles of sugar beet. Inoculation with F. acutatum did not result in any disease symptoms. The sugar beet disease caused by F. secorum is named Fusarium yellowing decline. Since Fusarium yellowing decline incidence has been increasing in the Red River Valley, disease management options are discussed.

Comparative Pathogenicity and Virulence of Fusarium Species on Sugar Beet.

In all, 98 isolates of three Fusarium spp. (18 Fusarium oxysporum, 30 F. graminearum, and 50 Fusarium sp. nov.) obtained from sugar beet in Minnesota were characterized for pathogenicity and virulence on sugar beet in the greenhouse by a bare-root inoculation method. Among the 98 isolates tested, 80% of isolates were pathogenic: 83% of the F. oxysporum isolates, 57% of the F. graminearum isolates, and 92% of the Fusarium sp. nov. isolates. Symptoms varied from slight to moderate wilting of the foliage, interveinal chlorosis and necrosis, and vascular discoloration of the taproot without any external root symptoms. Among the pathogenic isolates, 14% were highly virulent and 12% were moderately virulent. Most of the highly virulent isolates (91%) and moderately virulent isolates (89%) were Fusarium sp. nov. All pathogenic isolates of F. graminearum and most pathogenic isolates (87%) of F. oxysporum were less virulent. In general, more-virulent isolates induced first foliar symptoms earlier compared with less-virulent isolates. This study indicates that both F. oxysporum and Fusarium sp. nov. should be used in greenhouse and be present in field studies used for screening and developing sugar beet cultivars resistant to Fusarium yellows complex for Minnesota and North Dakota.

Efficacy of Variable Tetraconazole Rates Against Cercospora beticola Isolates with Differing In Vitro Sensitivities to DMI Fungicides.

Cercospora leaf spot (CLS) of sugar beet is caused by the fungus Cercospora beticola. CLS management practices include the application of the sterol demethylation inhibitor (DMI) fungicides tetraconazole, difenoconazole, and prothioconazole. Evaluating resistance to DMIs is a major focus for CLS fungicide resistance management. Isolates were collected in 1997 and 1998 (baseline sensitivity to tetraconazole, prothioconazole, or difenoconazole) and 2007 through 2010 from the major sugar-beet-growing regions of Minnesota and North Dakota and assessed for in vitro sensitivity to two or three DMI fungicides. Most (47%) isolates collected in 1997-98 exhibited 50% effective concentration (EC50) values for tetraconazole of <0.01 µg ml-1, whereas no isolates could be found in this EC50 range in 2010. Since 2007, annual median and mean tetraconazole EC50 values have generally been increasing, and the frequency of isolates with EC50 values >0.11 µg ml-1 increased from 2008 to 2010. In contrast, the frequency of isolates with EC50 values for prothioconazole of >1.0 µg ml-1 has been decreasing since 2007. Annual median difenoconazole EC50 values appears to be stable, although annual mean EC50 values generally have been increasing for this fungicide. Although EC50 values are important for gauging fungicide sensitivity trends, a rigorous comparison of the relationship between in vitro EC50 values and loss of fungicide efficacy in planta has not been conducted for C. beticola. To explore this, 12 isolates exhibiting a wide range of tetraconazole EC50 values were inoculated to sugar beet but no tetraconazole was applied. No relationship was found between isolate EC50 value and disease severity. To assess whether EC50 values are related to fungicide efficacy in planta, sugar beet plants were sprayed with various dilutions of Eminent, the commercial formulation of tetraconazole, and subsequently inoculated with isolates that exhibited very low, medium, or high tetraconazole EC50 values. The high EC50 isolate caused significantly more disease than isolates with medium or very low EC50 values at the field application rate and most reduced rates. Because in vitro sensitivity testing is typically carried out with the active ingredient of the commercial fungicide, we investigated whether loss of disease control was the same for tetraconazole as for the commercial product Eminent. The high EC50 isolate caused more disease on plants treated with tetraconazole than Eminent but disease severity was not different between plants inoculated with the very low EC50 isolate.

Temperature, moisture, and fungicide effects in managing Rhizoctonia root and crown rot of sugar beet.

Rhizoctonia solani AG-2-2 is the causal agent of Rhizoctonia root and crown rot in sugar beet; however, recent increases in disease incidence and severity were grounds to reevaluate this pathosystem. To assess the capacity at which other anastomosis groups (AGs) are able to infect sugar beet, 15 AGs and intraspecific groups (ISGs) were tested for pathogenicity on resistant ('FC708 CMS') and susceptible ('Monohikari') seedlings and 10-week-old plants. Several AGs and ISGs were pathogenic on seedlings regardless of host resistance but only AG-2-2 IIIB and AG-2-2 IV caused significant disease on 10-week-old plants. Because fungicides need to be applied prior to infection for effective disease control, temperature and moisture parameters were assessed to identify potential thresholds that limit infection. Root and leaf disease indices were used to evaluate disease progression of AG-2-2 IIIB- and AG-2-2 IV-inoculated plants in controlled climate conditions of 7 to 22 growing degree days (GDDs) per day. Root disease ratings were positively correlated with increasing temperature of both ISGs, with maximum disease symptoms occurring at 22 GDDs/day. No disease symptoms were evident from either ISG at 10 GDDs/day but disease symptoms did occur in plants grown in growth chambers set to 11 GDDs/day. Using growth chambers adjusted to 22 GDDs/day, disease was evaluated at 25, 50, 75, and 100% moisture-holding capacity (MHC). Disease symptoms for each ISG were highest in soils with 75 and 100% MHC but disease still occurred at 25% MHC. Isolates were tested for their ability to cause disease at 1, 4, and 8 cm from the plant hypocotyl. Only AG-2-2 IIIB was able to cause disease symptoms at 8 cm during the evaluation period. In all experiments, isolates of AG-2-2 IIIB were found to be more aggressive than AG-2-2 IV. Using environmental parameters that we identified as the most conducive to disease development, azoxystrobin, prothioconazole, pyraclostrobin, difenoconazole/propiconazole, flutolanil, polyoxin D, and a water control were evaluated for their ability to suppress disease development by AG-2-2 IIIB and AG-2-2 IV 17 days after planting. Flutolanil, polyoxin-D, and azoxystrobin provided the highest level of disease suppression. Because R. solani AG-2-2 IIIB and AG-2-2 IV are affected by temperature and moisture, growers may be able to evaluate environmental parameters for optimization of fungicide application.