Larry G. Campbell

Genetic Variability for Mineral Concentration in the Forage of Jerusalem Artichoke Cultivars

SummaryOne of the potential uses of Jerusalem artichoke (Helianthus tuberosus L.) is as a forage crop. Information on inherent differences in forage nutritional quality is essential if the quality of the forage is to be improved through breeding. The objectives of this study were to determine the genotypic variability among and within forage of Jerusalem artichoke cultivars for the concentration of N, P, Ca, Mg, K and the Ca/P ratio at flowering, to determine if selection among and within cultivars is feasible, to estimate the magnitude of the genotype × environment interaction, and to examine the relationships among mineral concentrations in the forage. Ten cultivated Jerusalem artichoke cultivars grown in an irrigated field nursery at Bushland, TX were evaluated for N, P, Ca, Mg, K, and the Ca/P ratio in the forage at flowering over a 2-yr period. Cultivars, cultivar × year, and error variances were estimated to calculate the phenotypic variance. Estimates of the within-population variances were also determined. The adequacy of Jerusalem artichoke forage at flowering for maintenance of a ruminant animal was classified as follows: N, Ca, Mg, K as adequate, P inadequate, and the Ca/P ratio as excessive. There were genotypic differences among the ten cultivars for N, P, Ca, Mg, K, and the Ca/P ratio for both years and averaged across years. The magnitude of the genotypic variance components indicated that a substantial proportion of the total variation for these elements was due to cultivar, indicating the possibility of improving these elements. However, further studies on heritability and response to selection will be required before conclusions can be reached concerning the likelihood of successfully breeding for these traits.

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.

Postharvest Storage Losses Associated with Rhizomania in Sugar Beet

During storage of sugar beet, respiration and rots consume sucrose and produce invert sugar. Diseases that occur in the field can affect the magnitude of these losses. This research examines the storage of roots with rhizomania (caused by Beet necrotic yellow vein virus) and the effectiveness of rhizomania-resistant hybrids in reducing postharvest losses. Roots of susceptible hybrids from sites with rhizomania had respiration rates 30 days after harvest (DAH) that ranged from 0.68 to 2.79 mg of CO2 kg-1 h-1 higher than roots of the resistant hybrids. This difference ranged from 2.60 to 13.88 mg of CO2 kg-1 h-1 120 DAH. Roots of resistant hybrids from sites with rhizomania had 18 kg more sucrose per ton than roots from susceptible hybrids 30 DAH, with this difference increasing to 55 kg Mg-1 120 DAH. The invert sugar concentration of susceptible hybrids from sites with rhizomania ranged from 8.38 to 287 g per 100 g of sucrose higher than that for resistant hybrids 120 DAH. In contrast, differences between susceptible and resistant hybrids in respiration rate, sucrose loss, and invert sugar concentration in the absence of rhizomania were relatively small. Storage losses due to rhizomania can be minimized by planting resistant hybrids and processing roots from fields with rhizomania soon after harvest.

Effects of Aphanomyces Root Rot on Carbohydrate Impurities and Sucrose Extractability in Postharvest Sugar Beet

Sugar beet (Beta vulgaris) roots with rot caused by Aphanomyces cochlioides often are incorporated into storage piles even though effects of disease on processing properties are unknown. Roots with Aphanomyces root rot were harvested from six fields over 2 years. For each field, roots with similar disease symptoms were combined and assigned a root rot index (RRI) value (0 to 100; 0, no rot symptoms; 100, all roots severely rotted). After 20 or 120 days storage at 4°C and 95% relative humidity, concentrations of the major carbohydrate impurities that accumulate during storage and sucrose extractability were determined. Root rot affected carbohydrate impurity concentrations and sucrose extractability in direct relation to disease severity symptoms. Generally, roots with active and severe infection (RRI ≥ 85) exhibited elevated glucose and fructose concentrations 20 and 120 days after harvest (DAH), elevated raffinose concentration 120 DAH, and reduced sucrose extractability 20 and 120 DAH. Roots with minor or moderate disease symptoms (RRI 20 to 69), or damaged roots with no signs of active infection, had similar carbohydrate impurity concentrations and sucrose extractability after 20 and 120 days storage. Processing properties declined when RRIs exceeded 43, as determined by regression analysis, or when storage duration increased from 20 to 120 days. Results indicate that both disease severity and anticipated duration of storage be considered before Aphanomyces-infected roots are incorporated into storage piles.

Glycolysis Is Dynamic and Relates Closely to Respiration Rate in Stored Sugarbeet Roots

Although respiration is the principal cause of the loss of sucrose in postharvest sugarbeet (Beta vulgaris L.), the internal mechanisms that control root respiration rate are unknown. Available evidence, however, indicates that respiration rate is likely to be controlled by the availability of respiratory substrates, and glycolysis has a central role in generating these substrates. To determine glycolytic changes that occur in sugarbeet roots after harvest and to elucidate relationships between glycolysis and respiration, sugarbeet roots were stored for up to 60 days, during which activities of glycolytic enzymes and concentrations of glycolytic substrates, intermediates, cofactors, and products were determined. Respiration rate was also determined, and relationships between respiration rate and glycolytic enzymes and metabolites were evaluated. Glycolysis was highly variable during storage, with 10 of 14 glycolytic activities and 14 of 17 glycolytic metabolites significantly altered during storage. Changes in glycolytic enzyme activities and metabolites occurred throughout the 60 day storage period, but were greatest in the first 4 days after harvest. Positive relationships between changes in glycolytic enzyme activities and root respiration rate were abundant, with 10 of 14 enzyme activities elevated when root respiration was elevated and 9 glycolytic activities static during periods of unchanging respiration rate. Major roles for pyruvate kinase and phosphofructokinase in the regulation of postharvest sugarbeet root glycolysis were indicated based on changes in enzymatic activities and concentrations of their substrates and products. Additionally, a strong positive relationship between respiration rate and pyruvate kinase activity was found indicating that downstream TCA cycle enzymes were unlikely to regulate or restrict root respiration in a major way. Overall, these results establish that glycolysis is not static during sugarbeet root storage and that changes in glycolysis are closely related to changes in sugarbeet root respiration.

Sugar beet root maggot (Tetanops myopaeformis) genes modulated by resistant and susceptible interactions with Beta vulgaris.

Suppressive subtractive hybridization (SSH) is a powerful tool for global analysis of gene expression and has been used in our laboratory to identify sugar beet root genes responsive to feeding by the sugar beet root maggot (SBRM, Tetanops myopaeformis). We are currently focusing our studies on the identification of SBRM genes whose expression is modulated by interactions with resistant or susceptible sugar beet germplasm. PCR-select SSH was used to generate cDNA libraries enriched for SBRM genes after contact of the pest with a moderately resistant F1016 and a susceptible F1010 germplasm. SBRM larvae were starved for 72 h and then fed F1016 or F1010 roots. At 1, 6, 24, 48 and 72 h after infestation, 20 larvae were collected for each time point for further analysis. Three complete subtractions were conducted using pooled tissues from the five time points: SBRM fed on F1016 vs. unfed, SBRM on F1010 vs. unfed, and SBRM on F1016 vs. on F1010. Screening of differentially expressed SBRM genes is ongoing. Genes identified as being important in resistant or susceptible pest-plant interactions will be selected for further analyses. New insights into the molecular response elicited by SBRM in interactions with sugar beet roots will advance the development of novel approaches for more effective SBRM control.

An in vitro sugar beet root maggot (Tetanops myopaeformis) feeding assay

Sugar beet root maggot (SBRM, Tetanops myopaeformis ROder) is a serious pest of sugar beet (Beta vulgaris L.) in North America and Canada Currently, insecticides are the most efficacious measure for control of the insect Therefore, alternative control measures are being sought. An in vitro system was established to study interactions between sugar beet roots and SBRM. Sources of root material included hairy root cultures, 14-day-old seedlings and taproots from I-year-old greenhouse plants. Hairy root cultures were stained in 0.01 % saffranin or crystal violet and placed on petri plates with Yz strength 85 medium or water-moistened Whatrnan 3 filter paper or nylon membrane. Seedlings and taproots were placed on nylon membranes. To reduce contamination, benomyl (10 mg/I), cefotaxirne (300 mg/I) and carbenicillin ( 400 mg/I) were added to the plates. First, second and third instar SBRM, obtained either from eggs oflaboratory-reared flies or from soil samples collected from infested sugar beet fields, were placed on the roots. Evidence of SBRM feeding included severed roots and saffranin or crystal violet in the frass or intestinal tracts of insects. Some larvae survived for more than 50 days on the roots. This bioassay will be useful for rapid screening of newly developed SBRM resistant sugar beet germplasm, chemical control agents or biocontrol organisms. Insecticidal plant extracts and spores of a biocontrol fungus, Synglioc/adium tetanopsis, are currently being evaluated by this assay.