The Tobacco Trichome Exudate Z-abienol and Its Relationship With Plant Resistance to Phytophthora nicotianae.

In previous research, we discovered a favorable quantitative trait locus (QTL) in cigar tobacco cultivar 'Beinhart 1000' designated as Phn15.1, which provides a high level of partial resistance to the black shank disease caused by Phytophthora nicotianae. A very close genetic association was also found between Phn15.1 and the ability to biosynthesize Z-abienol, a labdanoid diterpene exuded by the trichomes onto above-ground plant parts, and that imparts flavor and aroma characteristics to Oriental and some cigar tobacco types. Because accumulation of Z-abienol is considered to be undesirable for cultivars of other tobacco types, we herein describe a series of experiments to gain insight on whether this close association is due to genetic linkage or pleiotropy. First, in an in vitro bioassay, we observed Z-abienol and related diterpenes to inhibit hyphal growth of P. nicotianae at concentrations between 0.01 and 100 ppm. Secondly, we field-tested transgenic versions of Beinhart 1000 carrying RNAi constructs for downregulating NtCPS2 or NtABS, two genes involved in the biosynthesis of Z-abienol. Thirdly, we also field tested a recombinant inbred line population segregating for a truncation mutation in NtCPS2 leading to an interrupted Z-abienol pathway. We observed no correlation between field resistance to P. nicotianae and the ability to accumulate Z-abienol in either the transgenic materials or the mapping population. Results suggest that, although Z-abienol may affect P. nicotianae when applied at high concentrations in in vitro assays, the compound has little effect on black shank disease development under natural field conditions. Thus, it should be possible to disassociate Phn15.1-mediated black shank resistance identified in cigar tobacco cultivar Beinhart 1000 from the ability to accumulate Z-abienol, an undesirable secondary metabolite for burley and flue-cured tobacco cultivars.

Modulation of the phenylacetic acid metabolic complex by quinic acid alters the disease-causing activity of Rhizoctonia solani on tomato.

The metabolic control of plant growth regulator production by the plant pathogenic fungus Rhizoctonia solani Kühn (teleomorph=Thanatephorus cucumeris (A.B. Frank) Donk) and consequences associated with the parasitic and saprobic activity of the fungus were investigated. Fourteen genetically distinct isolates of the fungus belonging to anastomosis groups (AG) AG-3, AG-4, and AG-1-IA were grown on Vogel's minimal medium N with and without the addition of a 25 mM quinic acid (QA) source of carbon. The effect of QA on fungal biomass was determined by measuring the dry wt of mycelia produced under each growth condition. QA stimulated growth of 13 of 14 isolates of R. solani examined. The production of phenylacetic acid (PAA) and the chemically related derivatives 2-hydroxy-PAA, 3-hydroxy-PAA, 4-hydroxy-PAA, and 3-methoxy-PAA on the two different media was compared by gas chromatography coupled with mass spectrometry (GC-MS). The presence of QA in the growth medium of R. solani altered the PAA production profile, limiting the conversion of PAA to derivative forms. The effect of QA on the ability of R. solani to cause disease was examined by inoculating tomato (Solanum lycopersicum L.) plants with 11 isolates of R. solani AG-3 grown on media with and without the addition of 25 mM QA. Mean percent survival of tomato plants inoculated with R. solani was significantly higher when the fungal inoculum was generated on growth medium containing QA. The results of this study support the hypotheses that utilization of QA by R. solani leads to reduced production of the plant growth regulators belonging to the PAA metabolic complex which can suppress plant disease development.

Impact of alleles at the Yellow Burley (Yb) loci and nitrogen fertilization rate on nitrogen utilization efficiency and tobacco-specific nitrosamine (TSNA) formation in air-cured tobacco.

Tobacco-specific nitrosamine (TSNA) formation in tobacco is influenced by alkaloid levels and the availability of nitrosating agents. Tobacco types differ in their potential for TSNA accumulation due to genetic, agronomic, and curing factors. Highest TSNA concentrations are typically measured in burley tobaccos. One of the main genetic differences between burley and all other tobacco types is that this tobacco type is homozygous for recessive mutant alleles at the Yellow Burley 1 (Yb(1)) and Yellow Burley 2 (Yb(2)) loci. In addition, burley tobacco is typically fertilized at higher nitrogen (N) rates than most other tobacco types. This study utilized nearly isogenic lines (NILs) differing for the presence of dominant or recessive alleles at the Yb(1) and Yb(2) loci to investigate the potential influence of genes at these loci on TSNA accumulation. Three pairs of NILs were evaluated at three different nitrogen fertilization rates for alkaloid levels, nitrogen physiology measures, and TSNA accumulation after air-curing. As previously observed by others, positive correlations were observed between N application rates and TSNA accumulation. Recessive alleles at Yb(1) and Yb(2) were associated with increased alkaloid levels, reduced nitrogen use efficiency, reduced nitrogen utilization efficiency, and increased leaf nitrate nitrogen (NO(3)-N). Acting together, these factors contributed to significantly greater TSNA levels in genotypes possessing the recessive alleles at these two loci relative to those carrying the dominant alleles. The chlorophyll-deficient phenotype conferred by the recessive yb(1) and yb(2) alleles probably contributes in a substantial way to increase available NO(3)-N during curing and, consequently, increased potential for TSNA formation.

Elucidating the role of the phenylacetic acid metabolic complex in the pathogenic activity of Rhizoctonia solani anastomosis group 3.

The soil fungus Rhizoctonia solani produces phytotoxic phenylacetic acid (PAA) and hydroxy (OH-) and methoxy (MeO-) derivatives of PAA. However, limited information is available on the specific role that these compounds play in the development of Rhizoctonia disease symptoms and concentration(s) required to induce a host response. Reports that PAA inhibits the growth of R. solani conflict with the established ability of the fungus to produce and metabolize PAA. Experiments were conducted to clarify the role of the PAA metabolic complex in Rhizoctonia disease. In this study the concentration of PAA and derivatives required to induce tomato root necrosis and stem canker, in the absence of the fungus, and the concentration that inhibits mycelial growth of R. solani were determined. The effect of exogenous PAA and derivatives of PAA on tomato seedling growth also was investigated. Growth of tomato seedlings in medium containing 0.1-7.5 mM PAA and derivatives induced necrosis of up to 85% of root system. Canker development resulted from injection of tomato seedling stems with 7.5 mM PAA, 3-OH-PAA, or 3-MeO-PAA. PAA in the growth medium reduced R. solani biomass, with 50% reduction observed at 7.5 mM. PAA, and derivatives were quantified from the culture medium of 14 isolates of R. solani belonging to three distinct anastomosis groups by GC-MS. The quantities ranged from below the limit of detection to 678 nM, below the concentrations experimentally determined to be phytotoxic. Correlation analyses revealed that isolates of R. solani that produced high PAA and derivatives in vitro also caused high mortality on tomato seedlings. The results of this investigation add to the body of evidence that the PAA metabolic complex is involved in Rhizoctonia disease development but do not indicate that production of these compounds is the primary or the only determinant of pathogenicity.

Analysis of CoQ10 in cultivated tobacco by a high-performance liquid chromatography-ultraviolet method.

Coenzyme Q (CoQ) is a naturally occurring lipid-soluble quinone that performs multiple functions in all living cells and has become a popular antioxidant supplement, a coadjuvant in the treatment of heart disease, and the object of study for treating neurodegenerative disorders. Although there are many tools for CoQ analysis of microbial and animal samples, there have been relatively few reports of methods for CoQ analysis of green plants. This work describes a method for the routine analysis of coenzyme Q(10) in green leaf tissue of cultivated Nicotiana tabacum (tobacco) using high-performance liquid chromatography (HPLC) with UV detection. The method was applied to the analysis of CoQ(10) in N. tabacum 'KY14' leaves at different stalk positions representing young lanceolate to senescing leaves, and it was found that CoQ(10) increased as leaf position changed down the stalk from 18.69 to 82.68 µg/g fw. The method was also used to observe CoQ(10) in N. tabacum 'NC55' and N. tabacum 'TN90LC' leaves over time, finding that CoQ(10) leaf content remained relatively stable from 3 to 6 weeks but increased in both cultivars at 8 weeks. This method will likely be useful in the analysis of CoQ(10) in the green leaves of other plant species.

Analysis of a Nicotiana tabacum L. genomic region controlling two leaf surface chemistry traits.

cis-Abienol and sucrose esters are Nicotiana tabacum leaf surface components that likely influence plant resistance to pests. Their breakdown products also contribute to flavor and aroma characteristics of certain tobacco types. Mapping of genes involved in the biosynthesis of these compounds could permit development of molecular-based tools for generating tobacco types with novel cured leaf chemistry profiles. A doubled haploid mapping population segregating for major genes (Abl and BMVSE) affecting the ability to accumulate cis-abienol and sucrose esters was generated and genotyped with a large set of microsatellite markers. The two genes were found to reside on chromosome A of the N. tabacum genome with a distance of 8.2 cM (centimorgans) between them. Seventeen microsatellite markers were also placed on this linkage group, several of which exhibited complete cosegregation with Abl and BMVSE. Results should aid breeding efforts focused on modification of this aspect of tobacco cured leaf chemistry.

Analysis of chlorothalonil and degradation products in soil and water by GC/MS and LC/MS.

We present a method using gas chromatography (GC) and liquid chromatography (LC) coupled to a mass selective detector to measure concentrations of the fungicide chlorothalonil and several of its metabolites in soil and water. The methods employed solid-phase extraction using a hydrophobic polymeric phase for the isolation of analytes. In lake water, average analyte recoveries ranged from 70% to 110%, with exception of pentachloronitrobenzene that gave low recoveries (23%). The method detection limits were determined to be in the range of 1 and 0.1microg l(-1) for the LC and GC methods, respectively. In soil samples, recoveries ranged from 80% to 95% for 4-hydroxy-2,5,6-trichloroisophthalonitrile (metabolite II) and 1,3-dicarbamoyl-2,4,5,6-tetrachlorobenzene (metabolite III). Limits of detection (LOD) were 0.05 and 0.02microg g(-1), respectively. Chlorothalonil and other metabolites were analyzed by GC giving recoveries ranging from 54% to 130% with LOD of 0.001-0.005microg g(-1).

Vitamer levels, stress response, enzyme activity, and gene regulation of Arabidopsis lines mutant in the pyridoxine/pyridoxamine 5'-phosphate oxidase (PDX3) and the pyridoxal kinase (SOS4) genes involved in the vitamin B6 salvage pathway.

PDX3 and SALT OVERLY SENSITIVE4 (SOS4), encoding pyridoxine/pyridoxamine 5'-phosphate oxidase and pyridoxal kinase, respectively, are the only known genes involved in the salvage pathway of pyridoxal 5'-phosphate in plants. In this study, we determined the phenotype, stress responses, vitamer levels, and regulation of the vitamin B(6) pathway genes in Arabidopsis (Arabidopsis thaliana) plants mutant in PDX3 and SOS4. sos4 mutant plants showed a distinct phenotype characterized by chlorosis and reduced plant size, as well as hypersensitivity to sucrose in addition to the previously noted NaCl sensitivity. This mutant had higher levels of pyridoxine, pyridoxamine, and pyridoxal 5'-phosphate than the wild type, reflected in an increase in total vitamin B(6) observed through HPLC analysis and yeast bioassay. The sos4 mutant showed increased activity of PDX3 as well as of the B(6) de novo pathway enzyme PDX1, correlating with increased total B(6) levels. Two independent lines with T-DNA insertions in the promoter region of PDX3 (pdx3-1 and pdx3-2) had decreased PDX3 activity. Both also had decreased activity of PDX1, which correlated with lower levels of total vitamin B(6) observed using the yeast bioassay; however, no differences were noted in levels of individual vitamers by HPLC analysis. Both pdx3 mutants showed growth reduction in vitro and in vivo as well as an inability to increase growth under high light conditions. Increased expression of salvage and some of the de novo pathway genes was observed in both the pdx3 and sos4 mutants. In all mutants, increased expression was more dramatic for the salvage pathway genes.

Changes over time in the allelochemical content of ten cultivars of rye (Secale cereale L.).

Published studies focused on characterizing the allelopathy-based weed suppression by rye cover crop mulch have provided varying and inconsistent estimates of weed suppression. Studies were initiated to examine several factors that could influence the weed suppressiveness of rye: kill date, cultivar, and soil fertility. Ten cultivars of rye were planted with four rates of nitrogen fertilization, and tissue from each of these treatment combinations was harvested three times during the growing season. Concentrations of a known rye allelochemical DIBOA (2,4-dihydroxy-1,4-(2H)benzoxazine-3-one) were quantified from the harvested rye tissue using high performance liquid chromatography (HPLC). Phytotoxicity observed from aqueous extracts of the harvested rye tissue correlated with the levels of DIBOA recovered in harvested tissue. The amount of DIBOA in rye tissue varied depending on harvest date and rye cultivar, but was generally lower with all cultivars when rye was harvested later in the season. However, the late maturing variety 'Wheeler' retained greater concentrations of DIBOA in comparison to other rye cultivars when harvested later in the season. The decline in DIBOA concentrations as rye matures, and the fact that many rye cultivars mature at different rates may help explain why estimates of weed suppression from allelopathic agents in rye have varied so widely in the literature.

Xanosporic acid, an intermediate in bacterial degradation of the fungal phototoxin cercosporin.

The red fungal perylenequinone phototoxin cercosporin is oxidized by Xanthomonas campestris pv zinniae to a non-toxic, unstable green metabolite xanosporic acid, identified via its lactone as 1,12-bis(2'R-hydroxypropyl)-4,9-dihydroxy-6,7-methylenedioxy-11-methoxy-3-oxaperylen-10H-10-one-2-carboxylic acid. Xanosporolactone was isolated in approximately 2:1 ratio of M:P atropisomers.