Combined Omics Approaches Reveal Distinct Mechanisms of Resistance and/or Susceptibility in Sugar Beet Double Haploid Genotypes at Early Stages of Beet Curly Top Virus Infection.

Sugar beet is susceptible to Beet curly top virus (BCTV), which significantly reduces yield and sugar production in the semi-arid growing regions worldwide. Sources of genetic resistance to BCTV is limited and control depends upon insecticide seed treatments with neonicotinoids. Through double haploid production and genetic selection, BCTV resistant breeding lines have been developed. Using BCTV resistant (R) [KDH13; Line 13 and KDH4-9; Line 4] and susceptible (S) [KDH19-17; Line 19] lines, beet leafhopper mediated natural infection, mRNA/sRNA sequencing, and metabolite analyses, potential mechanisms of resistance against the virus and vector were identified. At early infection stages (2- and 6-days post inoculation), examples of differentially expressed genes highly up-regulated in the 'R' lines (vs. 'S') included EL10Ac5g10437 (inhibitor of trypsin and hageman factor), EL10Ac6g14635 (jasmonate-induced protein), EL10Ac3g06016 (ribosome related), EL10Ac2g02812 (probable prolyl 4-hydroxylase 10), etc. Pathway enrichment analysis showed differentially expressed genes were predominantly involved with peroxisome, amino acids metabolism, fatty acid degradation, amino/nucleotide sugar metabolism, etc. Metabolite analysis revealed significantly higher amounts of specific isoflavonoid O-glycosides, flavonoid 8-C glycosides, triterpenoid, and iridoid-O-glycosides in the leaves of the 'R' lines (vs. 'S'). These data suggest that a combination of transcriptional regulation and production of putative antiviral metabolites might contribute to BCTV resistance. In addition, genome divergence among BCTV strains differentially affects the production of small non-coding RNAs (sncRNAs) and small peptides which may potentially affect pathogenicity and disease symptom development.

Leaf Bacteriome in Sugar Beet Shows Differential Response against Beet curly top virus during Resistant and Susceptible Interactions.

Beet curly top virus (BCTV) significantly reduces sugar beet yield in semi-arid production areas. Genetic resistance to BCTV is limited; therefore, identification of additional resistance-associated factors is highly desired. Using 16S rRNA sequencing and BCTV resistant (R) genotypes (KDH13, KDH4-9) along with a susceptible (S) genotype (KDH19-17), we investigated leaf bacteriome changes during BCTV post inoculation (pi). At day 6 (~6-week-old plants), Cyanobacteria were predominant (~90%); whereas, at week 4 (~10-week-old plants) Firmicutes (11-66%), Bacteroidetes (17-26%), and Verrucomicrobia (12-29%) were predominant phyla and genotype dependent. Both Bacteroidetes and Verrucomicrobia, increased post infection only in the R lines. The bacterial genera Brevibacillus increased at 6 dpi, and Akkermansia and Bacteroides at 4 wkpi in the R lines. Linear discriminant analysis effect size (LEfSe) identified potential biomarkers in the R vs. S lines. Functional profiling revealed bacterial enrichment associated with the TCA cycle, polyisoprenoid, and L-methionine biosynthesis pathways only in KDH4-9 at 6 dpi. At 4 wkpi, bacteria associated with tryptophan and palmitate biosynthesis in the R lines, and uridine monophosphate, phosphatidyl glycerol, and phospholipid biosynthesis in the S line, were enriched. Future characterization of bacterial genera with antiviral properties will help establish their use as biocontrol agents/biomarkers against BCTV.

Cell-Wall-Degrading Enzymes-Related Genes Originating from Rhizoctonia solani Increase Sugar Beet Root Damage in the Presence of Leuconostoc mesenteroides.

Sugar beet crown and root rot caused by Rhizoctonia solani is a major yield constraint. Root rot is highly increased when R. solani and Leuconostoc mesenteroides co-infect roots. We hypothesized that the absence of plant cell-wall-degrading enzymes in L. mesenteroides and their supply by R. solani during close contact, causes increased damage. In planta root inoculation with or without cell-wall-degrading enzymes showed greater rot when L. mesenteroides was combined with cellulase (22 mm rot), polygalacturonase (47 mm), and pectin lyase (57 mm) versus these enzymes (0-26 mm), R. solani (20 mm), and L. mesenteroides (13 mm) individually. Carbohydrate analysis revealed increased simpler carbohydrates (namely glucose + galactose, and fructose) in the infected roots versus mock control, possibly due to the degradation of complex cell wall carbohydrates. Expression of R. solani cellulase, polygalacturonase, and pectin lyase genes during root infection corroborated well with the enzyme data. Global mRNAseq analysis identified candidate genes and highly co-expressed gene modules in all three organisms that might be critical in host plant defense and pathogenesis. Targeting R. solani cell-wall-degrading enzymes in the future could be an effective strategy to mitigate root damage during its interaction with L. mesenteroides.

Incidence, Distribution, and Pathogenicity of Fungi Causing Root Rot in Idaho Long-Term Sugar Beet Storage Piles.

Fungal rots in sugar beet roots held in long-term storage can lead to considerable sucrose loss but the incidence and distribution of fungal rots inside sugar beet piles and pathogenicity for some species is poorly understood. Thus, Idaho sugar beet held in five outdoor and two indoor piles in 2014 and 2015 were investigated. The root surface area covered by fungal growth and discolored and healthy tissue were assessed in nine 1-m2 areas per pile using a stratified random sampling design. Pathogenicity was evaluated indoors via plug inoculation in 2015 and 2016. Botrytis cinerea covered more root surface area inside indoor piles (6 to 22%) than outdoor piles (0 to 3%) (P < 0.0001). No trends were evident for the Athelia-like sp. (0 to 15%) and Penicillium-type spp. (0 to 8%). Penicillium-type isolates comprised the following species: 60% Penicillium expansum, 34% P. cellarum, 3% P. polonicum, and 3% Talaromyces rugulosus. Trace levels (<1% of root surface) of other fungi, including Cladosporium and Fusarium spp., were evident on roots and in isolations. Based on sample location in a pile, there were no trends or differences; however, two outdoor piles (OVP1 and OVP2) had more healthy tissue (90 to 96%) than other piles (28 to 80%) (P < 0.0001). When the pathogenicity tests were analyzed by species, all were significantly different from each other (P < 0.0001), except for P. polonicum and P. expansum: B. cinerea (61 mm of rot), P. polonicum (36 mm), P. expansum (35 mm), P. cellarum (28 mm), Athelia-like sp. (21 mm), T. rugulosus (0 mm; not different from check), and noninoculated check (0 mm). The OVP1 and OVP2 piles had negligible fungal growth on roots after more than 120 days of storage under ambient conditions, which indicates that acceptable storage can be achieved over this time period through covering piles with tarps and cooling with ventilation pipe.

Influence of Beet necrotic yellow vein virus and Freezing Temperatures on Sugar Beet Roots in Storage.

Rhizomania caused by Beet necrotic yellow vein virus (BNYVV) is a yield-limiting sugar beet disease that was observed to influence root resistance to freezing in storage. Thus, studies were conducted to gain a better understanding of the influence of BNYVV and freezing on sugar beet roots to improve pile management decisions. Roots from five commercial sugar beet cultivars (one susceptible and four resistant to BNYVV) were produced in fields under high and trace levels of rhizomania pressure and subjected to storage using five temperature regimes ranging from 0 to -4.4°C for 24 h. After cold treatment, eight-root samples were stored in a commercial indoor storage building (set point 1.1°C) for 50 days in 2014 and 57 days in 2015. Internal root temperature, frozen and discolored tissue, and moisture and sucrose loss were evaluated. The air temperature at 0, -1.1, and -2.2°C matched internal root temperature but internal root remained near -2.2°C when air temperature was dropped to -3.3 and -4.4°C. In a susceptible cultivar produced under high rhizomania pressure, the percentage of frozen tissue increased (P < 0.0001) from an average of 0 to 7% at 0, -1.1, and -2.2°C up to 16 to 63% at -3.3°C and 63 to 90% at -4.4°C, depending on year. Roots from the susceptible cultivar produced under low rhizomania pressure and those from the resistant cultivars from both fields only had elevated (P ≤ 0.05) frozen tissue at -4.4°C in 15 of 18 cultivar-year combinations. Frozen tissue was related to discolored tissue (r2 = 0.91), weight loss (r2 = 0.12 to 0.28), and sucrose reduction (r2 = 0.69 to 0.74). Consequently, BNYVV will not only lead to yield and sucrose loss in susceptible sugar beet cultivars but also to more frozen root tissue as temperatures drop below -2.2°C. Based on these observations, the air used to cool roots in nonfrozen sugar beet piles throughout the winter should not drop below -2.2°C to maximize sucrose retention.

A Novel Penicillium sp. Causes Rot in Stored Sugar Beet Roots in Idaho.

Penicillium vulpinum along with a number of other fungi can lead to rot of stored sugar beet roots. However, Penicillium isolates associated with necrotic lesions on roots from a recent sugar beet storage study were determined to be different from P. vulpinum and other recognized Penicillium species. Phylogenies based on sequencing of the internal transcribed spacer (ITS)-5.8S, ß-tubulin (BenA), and RNA polymerase II second largest subunit (RPB2) DNA regions indicate that these isolates are novel, but most closely related to the following Penicillium spp. in the section Fasiculata: P. aurantiogriseum, P. camemberti, and P. freii. Macro- and micromorphological data also support designating these isolates as a new species for which we propose the name, Penicillium cellarum sp. nov. Inoculation studies with the P. cellarum isolates on roots of the commercial sugar beet cultivar B-7 led to the formation of necrotic lesions 23 to 25 mm in diameter after 86 days in storage. These lesions were similar to those observed on sugar beet roots in commercial storage piles. These data indicate that P. cellarum is a pathogen which can cause root rot in stored sugar beet roots.

Beet curly top virus Strains Associated with Sugar Beet in Idaho, Oregon, and a Western U.S. Collection.

Curly top of sugar beet is a serious, yield-limiting disease in semiarid production areas caused by Beet curly top virus (BCTV) and transmitted by the beet leafhopper. One of the primary means of control for BCTV in sugar beet is host resistance but effectiveness of resistance can vary among BCTV strains. Strain prevalence among BCTV populations was last investigated in Idaho and Oregon during a 2006-to-2007 collection but changes in disease severity suggested a need for reevaluation. Therefore, 406 leaf samples symptomatic for curly top were collected from sugar beet plants in commercial sugar beet fields in Idaho and Oregon from 2012 to 2015. DNA was isolated and BCTV strain composition was investigated based on polymerase chain reaction assays with strain-specific primers for the Severe (Svr) and California/Logan (CA/Logan) strains and primers that amplified a group of Worland (Wor)-like strains. The BCTV strain distribution averaged 2% Svr, 30% CA/Logan, and 87% Wor-like (16% had mixed infections), which differed from the previously published 2006-to-2007 collection (87% Svr, 7% CA/Logan, and 60% Wor-like; 59% mixed infections) based on a contingency test (P < 0.0001). Whole-genome sequencing (GenBank accessions KT276895 to KT276920 and KX867015 to KX867057) with overlapping primers found that the Wor-like strains included Wor, Colorado and a previously undescribed strain designated Kimberly1. Results confirm a shift from Svr being one of the dominant BCTV strains in commercial sugar beet fields in 2006 to 2007 to becoming undetectable at times during recent years.

Leuconostoc spp. Associated with Root Rot in Sugar Beet and Their Interaction with Rhizoctonia solani.

Rhizoctonia root and crown rot is an important disease problem in sugar beet caused by Rhizoctonia solani and also shown to be associated with Leuconostoc spp. Initial Leuconostoc studies were conducted with only a few isolates and the relationship of Leuconostoc with R. solani is poorly understood; therefore, a more thorough investigation was conducted. In total, 203 Leuconostoc isolates were collected from recently harvested sugar beet roots in southern Idaho and southeastern Oregon during 2010 and 2012: 88 and 85% Leuconostoc mesenteroides, 6 and 15% L. pseudomesenteroides, 2 and 0% L. kimchi, and 4 and 0% unrecognized Leuconostoc spp., respectively. Based on 16S ribosomal RNA sequencing, haplotype 11 (L. mesenteroides isolates) comprised 68 to 70% of the isolates in both years. In pathogenicity field studies with commercial sugar beet 'B-7', all Leuconostoc isolates caused more rot (P < 0.0001; α = 0.05) when combined with R. solani than when inoculated alone in both years. Also, 46 of the 52 combination treatments over the 2 years had significantly more rot (P < 0.0001; α = 0.05) than the fungal check. The data support the conclusion that a synergistic interaction leads to more rot when both Leuconostoc spp. and R. solani are present in sugar beet roots.

Characterization of a Basidiomycete fungus from stored sugar beet roots.

Eighteen isolates from sugar beet roots associated with an unknown etiology were characterized based on observations of morphological characters, hyphal growth at 4-28 C, production of phenol oxidases and sequence analysis of internal transcribed spacer (ITS) and large subunit (LSU) regions of the ribosomal DNA (rDNA). The isolates did not produce asexual or sexual spores, had binucleate hyphal cells with clamp connections, grew 4-22 C with estimated optimal growth at 14.5 C and formed a dark brown pigment on potato dextrose or malt extract agar amended with 0.5% tannic acid. Color changes observed when solutions of gum guiac, guiacol and syringaldzine were applied directly to mycelium grown on these media indicated that all isolates produced phenol oxidases. Sequences of ITS and LSU regions on the rDNA gene from 15 isolates were 99.2-100% identical, and analysis of sequence data with maximum likelihood and maximum parsimony suggest that the isolates from sugar beet roots are phylogenetically related to Athelia bombacina, Granulobasidium vellereum and Cyphella digitalis. High statistical support for both loci under different criteria confirmed that Athelia bombacina was consistently the closest known relative to the sugar beet isolates. Additional taxonomic investigations are needed before species can be clarified and designated for these isolates.