Characterizing and Mapping Resistance in Synthetic-Derived Wheat to Rhizoctonia Root Rot in a Green Bridge Environment.

Root rot caused by Rhizoctonia spp. is an economically important soilborne disease of spring-planted wheat in growing regions of the Pacific Northwest (PNW). The main method of controlling the disease currently is through tillage, which deters farmers from adopting the benefits of minimal tillage. Genetic resistance to this disease would provide an economic and environmentally sustainable resource for farmers. In this study, a collection of synthetic-derived genotypes was screened in high-inoculum and low-inoculum field environments. Six genotypes were found to have varying levels of resistance and tolerance to Rhizoctonia root rot. One of the lines, SPBC-3104 ('Vorobey'), exhibited good tolerance in the field and was crossed to susceptible PNW-adapted 'Louise' to examine the inheritance of the trait. A population of 190 BC1-derived recombinant inbred lines was assessed in two field green bridge environments and in soils artificially infested with Rhizoctonia solani AG8. Genotyping by sequencing and composite interval mapping identified three quantitative trait loci (QTL) controlling tolerance. Beneficial alleles of all three QTL were contributed by the synthetic-derived genotype SPCB-3104.

Development of PCR Assays for Diagnosis and Detection of the Pathogens Phacidiopycnis washingtonensis and Sphaeropsis pyriputrescens in Apple Fruit.

Speck rot caused by Phacidiopycnis washingtonensis and Sphaeropsis rot caused by Sphaeropsis pyriputrescens are two recently reported postharvest diseases of apple. Infection by these two pathogens occurs in the orchard but remains latent before harvest. Symptoms develop after harvest and are similar to those of gray mold caused by Botrytis cinerea. Accurate diagnosis of these diseases is important during the fruit inspection process, particularly in the instance of fruit destined for export. Early near-harvest detection of latent infections in apple fruit is an important step to implement relevant pre- and postharvest measures for disease control. The aim of this study was to develop polymerase chain reaction (PCR) assays for diagnosis and early detection of latent infections of apple fruit by P. washingtonensis and S. pyriputrescens. Species-specific primers based on the ribosomal DNA internal transcribed spacer region were designed for use in PCR assays. Conventional and real-time PCR assays were developed and validated using fruit inoculated with P. washingtonensis, S. pyriputrescens, or B. cinerea and compared with identifications using traditional isolation-based assays. For wound-inoculated fruit, the PCR assays consistently provided the correct identification of the pathogen used as the inoculant in 6 h of processing time, compared with 5 to 6 days using culture-based methods. Real-time PCR assays effectively detected latent infections in symptomless stem and calyx tissues of fruit that were inoculated with the pathogens in the orchard during the growing season. The PCR assays provide a rapid, accurate method for diagnosis and early detection of these diseases.

Development and Evaluation of a TaqMan Real-Time PCR Assay for Fusarium oxysporum f. sp. spinaciae.

Fusarium oxysporum f. sp. spinaciae, causal agent of spinach Fusarium wilt, is an important soilborne pathogen in many areas of the world where spinach is grown. The pathogen is persistent in acid soils of maritime western Oregon and Washington, the only region of the United States suitable for commercial spinach seed production. A TaqMan real-time polymerase chain reaction (PCR) assay was developed for rapid identification and quantification of the pathogen, based on sequencing the intergenic spacer (IGS) region of rDNA of isolates of the pathogen. A guanine single-nucleotide polymorphism (G SNP) was detected in the IGS sequences of 36 geographically diverse isolates of F. oxysporum f. sp. spinaciae but not in the sequences of 64 isolates representing other formae speciales and 33 isolates representing other fungal species or genera. The SNP was used to develop a probe for a real-time PCR assay. The real-time PCR assay detected F. oxysporum f. sp. spinaciae at 3-14,056 CFU/g of soil in 82 soil samples collected over 3 years from naturally infested spinach seed production sites in western Washington, although a reliable detection limit of the assay was determined to be 11 CFU/g of soil. A significant (P < 0.05), positive correlation between enumeration of F. oxysporum on Komada's agar and quantification of the pathogen using the TaqMan assay was observed in a comparison of 82 soil samples. Correlations between pathogen DNA levels, Fusarium wilt severity ratings, and spinach biomass were significantly positive for one set of naturally infested soils but not between pathogen DNA levels, wilt incidence ratings, and spinach biomass for other soil samples, suggesting that soilborne pathogen population is not the sole determinant of spinach Fusarium wilt incidence or severity. The presence of the G SNP detected in one isolate of each of F. oxysporum ff. spp. lageneriae, lilii, melongenae, and raphani and reaction of the real-time PCR assay with 16 of 22 nonpathogenic isolates of F. oxysporum associated with spinach plants or soil in which spinach had been grown potentially limits the application of this assay. Nonetheless, because all isolates of F. oxysporum f. sp. spinaciae tested positive with the real-time PCR assay, the assay may provide a valuable means of screening for resistance to Fusarium wilt by quantifying development of the pathogen in spinach plants inoculated with the pathogen.

Identification and quantification of Rhizoctonia solani and R. oryzae using real-time polymerase chain reaction.

Rhizoctonia solani and R. oryzae are the principal causal agents of Rhizoctonia root rot in dryland cereal production systems of the Pacific Northwest. To facilitate the identification and quantification of these pathogens in agricultural samples, we developed SYBR Green I-based real-time quantitative-polymerase chain reaction (Q-PCR) assays specific to internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA of R. solani and R. oryzae. The assays were diagnostic for R. solani AG-2-1, AG-8, and AG-10, three genotypes of R. oryzae, and an AG-I-like binucleate Rhizoctonia species. Quantification was reproducible at or below a cycle threshold (Ct) of 33, or 2 to 10 fg of mycelial DNA from cultured fungi, 200 to 500 fg of pathogen DNA from root extracts, and 20 to 50 fg of pathogen DNA from soil extracts. However, pathogen DNA could be specifically detected in all types of extracts at about 100-fold below the quantification levels. Soils from Ritzville, WA, showing acute Rhizoctonia bare patch harbored 9.4 to 780 pg of R. solani AG-8 DNA per gram of soil.. Blastn, primer-template duplex stability, and phylogenetic analyses predicted that the Q-PCR assays will be diagnostic for isolates from Australia, Israel, Japan, and other countries.

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction.

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.

First Report of Damping-Off of Canola Caused by Rhizoctonia solani AG 2-1 in Washington State.

In early September 2003, winter canola (Brassica napus L) cv. Inca was direct seeded into plots previously cropped with spring barley at the Washington State University Dryland Research Station at Lind, WA. Before planting, the plots received 80 mm of water by sprinkler irrigation, and 2 weeks later, volunteer barley was killed with Paraquat contact herbicide. In late September, 3 weeks after planting, canola seedlings exhibited postemergence damping-off and lesions on the hypocotyls, resulting in significant stand reductions. Rhizoctonia solani was isolated from infected hypocotyls using water agar amended with chloramphenicol (100 µg/ml). Cultures on potato dextrose agar produced dark brown colonies with dark brown microsclerotia. Three isolates were grown on autoclaved oat seed for 3 weeks in 1-liter Erlenmeyer flasks at 22°C, and colonized seed was air dried in a laminar flow hood, ground in a coffee grinder, and added to a Thatuna silt loam at 1% (w/w). The infested soil was placed into 4- × 20.5-cm plastic tubes and planted with five canola seeds per tube, five tubes per isolate. In the control treatment, soil was not infested. Plants were grown in a temperature-controlled room in a greenhouse at 16°C, 12-h light/dark. Isolates caused pre- and postemergence damping-off after 1 week, and the surviving seedlings had significantly less plant height and dry weight. Isolates were identified as AG 2-1 by pairing cultures with AG 8, 2-1, and 10 on agar-coated slides (1). Selected isolates were also identified as AG 2-1 by sequencing of the ITS 1 and 2 regions of the rDNA and matching them to sequences in GenBank. On a farm north of Pullman, WA in 2004, R. solani was isolated from soil in spring and winter wheat fields using a toothpick baiting method (2). R. solani was found primarily from sites previously cropped with winter and spring canola. These isolates were identified as AG 2-1 and five isolates were tested in the greenhouse, as described above, on canola (cv. Inca), lentil (Lens culinaris Medik. cv. Merrit), wheat (Triticum aestivum L. cv. Madsen), barley (Hordeum vulgare L. cv. Baronesse), pea (Pisum sativum L. cv. Stirling), and chickpea (Cicer arietinum L. cv. Sierra). Three of five isolates significantly reduced emergence of canola, and all isolates significantly reduced dry weight of canola seedlings and caused lesions on hypocotyls. None of the isolates reduced emergence of the other crops. All isolates reduced the dry weight of pea and three isolates reduced plant height. None of the isolates reduced the dry weight of lentil, chickpea, wheat, or barley. One of the isolates was also tested on Arabidopsis thaliana and found to be pathogenic. R. solani AG 2-1 has been reported as an important pathogen on canola in Canada and Australia, but has not been reported from the Pacific Northwest of the United States. R. solani AG 2-1 is also pathogenic on rapeseed, mustard, and subterranean clover and has been isolated from wheat, sugar beets, and potato (3). Canola is a minor rotation crop in cereal-based cropping systems in eastern Washington (1,600 ha in 2005), but there is increasing interest in this oilseed crop for biodiesel production. However, R. solani AG 2-1 may reduce stands and yield of canola. References: (1) W. C. Kronland and M. E. Stanghellini. Phytopathology 78:820, 1988. (2) T. C. Paulitz and K. L. Schroeder. Plant Dis.89:767, 2005. (3) B. Sneh et al. Identification of Rhizoctonia species. The American Phytopathological Society. St. Paul, MN, 1991.

Engineering deoxynivalenol metabolism in wheat through the expression of a fungal trichothecene acetyltransferase gene.

Fusarium head blight occurs in cereals throughout the world and is especially important in humid growing regions. Fusarium head blight (FHB) has re-emerged as a major disease of wheat and barley in the U.S. and Canada since 1993. The primary causal agents of FHB, Fusarium graminearum and Fusarium culmorum, can produce deoxynivalenol (DON), a trichothecene mycotoxin that enhances disease severity and poses a health hazard to humans and monogastric animals. To reduce the effects of DON on wheat, we have introduced FsTRI101, a Fusarium sporotrichioides gene formerly known as TriR, into the regenerable cultivar Bobwhite. TRI101 encodes an enzyme that transfers an acetyl moiety to the C3 hydroxyl group of trichothecenes. Four different transgenic plants carrying the FsTRI101 gene were identified. Although expression levels varied among the four lines, all of them accumulated FsTRI101 transcripts in endosperm and glume. TRI101-encoded acetyltransferase activity was detected in endosperm extracts of a single plant that accumulated FsTRI101 mRNA. Greenhouse resistance tests indicated that the accumulation of FsTRI101-encoded acetyltransferase in this plant confers partial protection against the spread of F. graminearum in inoculated wheat heads (spikes).

Dg93, a nodule-abundant mRNA of Datisca glomerata with homology to a soybean early nodulin gene.

We have isolated a 590-bp full-length cDNA clone designated Dg93, an mRNA that is highly expressed in symbiotic root nodules of the actinorhizal host Datisca glomerata. Dg93 mRNA encodes a deduced polypeptide of 105 amino acids with significant identity (74%) to the soybean (Glycine max) early nodulin (ENOD) gene GmENOD93 (Kouchi and Hata, 1993). Dg93 mRNA is abundant in nodules at 4 weeks post inoculation, the earliest time assayed, and steady-state mRNA levels remain elevated 11 weeks after inoculation. Spatial patterns of Dg93 mRNA expression are complex, with transcript accumulation in the nodule lobe meristem, early infection zone, periderm, and cells of the vascular cylinder, but not in the surrounding uninfected cortical cells. Dg93 is encoded by a small gene family in D. glomerata. To our knowledge, this is the first report of a gene from an actinorhizal host that is expressed in the nodule meristem and that shares sequence homology with an early nodulin gene from a legume.

Symbiotic root nodules of the actinorhizal plant Datisca glomerata express Rubisco activase mRNA.

N2-fixing symbiotic root nodules of the actinorhizal host Datisca glomerata express Dgrca (D. glomerata Rubisco activase) mRNA, a transcript usually associated with photosynthetic organs or tissues. In northern blots a mature, 1700-nucleotide Dgrca mRNA was detected in green plant organs (leaves, flowers, and developing fruits) and in nodules but was not detected in roots. A second message of 3000 nucleotides was observed only in nodules. Both size classes of transcripts were polyadenylated. The larger transcript was 2- to 5-fold more abundant than the mature mRNA; it was hybridized to an intronic probe, indicating that a stable, incompletely spliced transcript was accumulating. Treatment with light on excised nodules did not alter the relative abundance of the two species. In in situ hybridizations the Dgrca message was expressed intensely in the nuclei of infected cells. The Dgrca transcripts also accumulated at lower levels in uninfected cortical cells adjacent to the periderm and the vascular cylinder. mRNA encoding the large subunit of Rubisco (DgrbcL) was abundant in mature infected cells and in the amyloplast-rich sheath of uninfected cortical cells lying between the infected cells and nodule periderm. The proteins Rubisco activase, Rubisco, and the 33-kD O2-evolving complex subunit did not accumulate to detectable levels, indicating that a functional photosynthetic apparatus was not prevalent in nodule tissue. Signals or factors required for the transcription of Dgrca appeared to be present in nodules, but efficient splicing and translation of the message were not observed in Frankia-infected tissue where transcript accumulation was highest.

A transgenic mutant of Lactuca sativa (lettuce) with a T-DNA tightly linked to loss of downy mildew resistance.

One hundred and ninety-two independent primary transformants of lettuce cv. Diana were obtained by co-cultivation with Agrobacterium tumefaciens carrying constructs containing maize Ac transposase and Ds. R2 families were screened for mutations at four genes (Dm) for resistance to downy mildew. One family, designated dm3t524, had lost resistance to an isolate of Bremia lactucae expressing the avirulence gene Avr3. Loss of resistance segregated as a single recessive allele of Dm3. The mutation was not due to a large deletion as all molecular markers flanking Dm3 were present. Loss of Dm3 activity co-segregated with a T-DNA from which Ds had excised. Genomic DNA flanking the right border of this T-DNA was isolated by inverse polymerase chain reaction. This genomic sequence was present in four to five copies in wild-type cv. Diana. One copy was missing in all eight deletion mutants of Dm3 and altered in dm3t524, indicating tight physical linkage to Dm3. Three open reading frames (ORFs) occurred in a 6.6-kb region flanking the insertion site; however, expression of these ORFs was not detected. No similarities were detected between these ORFs and resistance genes cloned from other species. Transgenic complementation with 11-to 27-kb genomic fragments of Diana spanning the insertion site failed to restore Dm3 function to two ethyl methanesulfonate (EMS)-induced mutants of Dm3 or to cv. Cobham Green, which naturally lacks Dm3 activity. Therefore, either the T-DNA inserted extremely close to, but not within, Dm3 and the mutation may have been caused by secondary movement of Ds, or Dm3 activity is encoded by a gene extending beyond the fragments used for complementation.

Molecular analysis of irradiation-induced and spontaneous deletion mutants at a disease resistance locus in Lactuca sativa.

The major cluster of disease resistance genes in lettuce (Lactuca sativa) contains at least nine downy mildew resistance genes (Dm) spanning a genetic distance of 20cM and a physical distance of at least 6 Mb. Nine molecular markers that were genetically tightly linked to Dm3 were used to analyze nine independent deletion mutants and construct a map of the region surrounding Dm3. This analysis identified a linear order of deletion breakpoints and markers along the chromosome. There was no evidence for chromosomal rearrangements associated with the deletions. The region is not highly recombinagenic and the deletion breakpoints provided greater genetic resolution than meiotic recombinants. The region contains a mixture of high-and low-copy number sequences; no single-copy sequences were detected. Three markers hybridized to low-copy-number families of sequences that are duplicated predominantly close to Dm3. This was not true for sequences related to the triose-phosphate isomerase gene; these had been shown previously to be linked to Dm3, as well as to two independent clusters of Dm genes, and elsewhere in the genome. Two spontaneous mutants of Dm3 were identified; several markers flanking Dm3 are absent in one of these two mutants. The stability of the Dm3 region was also studied by analyzing the genotypes of diverse related cultivars. The 1.5 Mb region surrounding Dm3 has remained stable through many generations of breeding with and without selection for Dm3 activity.

Mutants of downy mildew resistance in Lactuca sativa (lettuce).

As part of our investigation of disease resistance in lettuce, we generated mutants that have lost resistance to Bremia lactucae, the casual fungus of downy mildew. Using a rapid and reliable screen, we identified 16 distinct mutants of Latuca sativa that have lost activity of one of four different downy mildew resistance genes (Dm). In all mutants, only a single Dm specificity was affected. Genetic analysis indicated that the lesions segregated as single, recessive mutations at the Dm loci. Dm3 was inactivated in nine of the mutants. One of five Dm 1 mutants was selected from a population of untreated seeds and therefore carried a spontaneous mutation. All other Dm1, Dm3, Dm5/8 and Dm7 mutants were derived from gamma- or fast neutron-irradiated seed. In two separate Dm 1 mutants and in each of the eight Dm3 mutants analyzed, at least one closely linked molecular marker was absent. Also, high molecular weight genomic DNA fragments that hybridized to a tightly linked molecular marker in wild type were either missing entirely or were truncated in two of the Dm3 mutants, providing additional evidence that deletions had occurred in these mutants. Absence of mutations at loci epistatic to the Dm genes suggested that such loci were either members of multigene families, were critical for plant survival, or encoded components of duplicated pathways for resistance; alternatively, the genes determining downy mildew resistance might be limited to the Dm loci.

Analysis of genes negatively regulated by phytochrome action in Lemna gibba and identification of a promoter region required for phytochrome responsiveness.

As a step to understanding how the photoreceptor phytochrome acts to change the transcription of specific nuclear genes in Lemna gibba, we wish to compare promoter elements involved in negative regulation by phytochrome with those involved in positive regulation. We have isolated three genes negatively regulated by phytochrome, designated NR (negatively phytochrome regulated) genes (P.A. Okubara, E.M. Tobin [1991] Plant Physiol 96:1237-1245), and we have now sequenced two of these. The promoters of both contain some sequence motifs that are identical with motifs from other genes. We used a transient assay in L. gibba to demonstrate that approximately 1.7 kb pairs of the NPR1 promoter and 1.1 kb pairs of the NPR2 promoter could confer negative phytochrome regulation to a luciferase reporter gene. Deletion analysis of the NPR2 promoter showed that sequences between -208 and -82 from the transcription start were necessary for negative phytochrome regulation. However, this region was not sufficient to confer negative regulation by phytochrome to another promoter. Additionally, we noted that this region showed no similarity to a region identified as important for the negative regulation of the oat phyA promoter (W.B. Bruce, X.-W. Deng, P.H. Quail [1991] EMBO J 10:3015-3024), but it does contain a sequence element found in several other kinds of genes, including ones positively regulated by phytochrome. The deduced amino acid sequences of NPR1 and NPR2 were found to share similarities with many abscisic acid-induced or seed-abundant proteins. Thus, these genes, like other phytochrome-regulated genes, might respond to multiple regulatory signals.

Isolation and Characterization of Three Genes Negatively Regulated by Phytochrome Action in Lemna gibba.

We have isolated three distinct cDNA clones from Lemna gibba representing mRNAs that increase in abundance during dark treatment. All three mRNAs showed reduced expression in response to red or white light. These mRNAs range from approximately 680 to 800 nucleotides in length and thus encode relatively small proteins (maximum relative molecular weight 17,000 to 19,000). The genes corresponding to these dark-abundant mRNAs are designated NPR (negatively phytochrome regulated) 1, 2, and 3. Differences in the rapidity of mRNA accumulation during dark treatment were observed for each of the genes in both mature green plants and in etiolated plants. Differences in accumulation pattern were also observed in etiolated plants, depending on whether the plants received a far-red light treatment prior to darkness. Transcription of all three genes, assayed in nuclei isolated from either green or etiolated plants, increased during dark treatment. In etiolated plants, a single 2 minute red light treatment caused a detectable decrease in the transcription of the genes after the dark treatment, and 10 minutes of far-red light given immediately after the red light resulted in a reversal of the effect of red light. Additionally, treatment of the plants with far-red light prior to darkness resulted in greater rates of transcription of the NPR genes. Therefore, we conclude that phytochrome action results in decreased transcription of these NPR genes. Each of the NPR mRNAs are encoded by one to two genes.

Characterization of a negatively light-regulated mRNA from Lemna gibba.

We have isolated a cDNA clone, called pLg106, which corresponds to a negatively light-regulated mRNA in the aquatic monocot Lemna gibba. Lg106 mRNA is more abundant in dark-treated plants than in plants grown under continuous white light. The levels of Lg106 mRNA reached a maximum 12-24 hours after plants had been placed in the dark. In vitro-labelled transcripts from nuclei isolated from white light-grown and darktreated Lemna hybridized equally to Lg106 DNA, indicating that transcription of the Lg106 gene was similar in both white light and darkness. Therefore, the higher level of Lg106 mRNA in the dark was not attributable to a change in transcription. We suggest that the stability of Lg106 mRNA is affected by changes in illumination. The steady-state level of Lg106 did not appear to be under phytochrome regulation, as the abundance of Lg106 mRNA from plants grown in intermittent red light or intermittent red followed immediately by far-red light was not significantly different from that of dark-treated plants. The size of the mRNA was estimated to be 650-700 nt. Genomic Southern blots indicated the presence of a single gene for Lg106. The Lg106 cDNA was sequenced and examined for similarities to nucleotide sequences in GenBank.

Factors affecting the level of kanamycin resistance in transformed sunflower cells.

A 230 base pair DNA segment containing the sequences 5' to the 700 to 750 nucleotide (nt) transcript 7' (ORF 3; RF Barker, KB Idler, DV Thompson, JD Kemp 1983 Plant Mol Biol 2: 335-350) of the octopine tumor inducing plasmid pTiA6 has been isolated. This region has (a) 180 base pairs of DNA upstream of the TATA box, (b) the start of RNA synthesis, and (c) the entire 5' untranslated region of the gene. We have fused this presumed promoter fragment to the neomycin phosphotransferase II (NPTII) gene from Tn5 in a plant expression cassette. After recombination into a tumor inducing plasmid delivery plasmid, this cassette confers selectable kanamycin resistance to transformed sunflower cells. Removal of the out-of-frame ATG in the 5' leader sequence of the NPTII gene by two different modifications increased both the levels of NPTII enzyme activity and the ID(50) for kanamycin in the tumor cells. The promoter region of the transcript 7 gene gives levels of kanamycin resistance equivalent to the nopaline synthase promoter and octopine synthase promoter when used in the same constructions and assayed in the same tissues.

Reproduction in mice: liver enlargement in mice during pregnancy and lactation.

The mechanism of liver enlargement during pregnancy was investigated in the C57BL/6J strain of mice. The C57BL/6J female exhibited a two-fold increase in liver mass during pregnancy. After the completion of lactation the size of the liver was reduced. Liver growth was accomplished with no increase in hepatocyte number and without an increase in total liver DNA content. During the early stages of liver expansion in pregnant females, DNA synthesis could be turned on by partial hepatectomy. However, during the last few days of gestation DNA synthesis and liver growth in response to partial hepatectomy were inhibited. During lactation this inhibition of growth was maintained, but inhibition of DNA synthesis was partially lifted. DNA synthesis and liver growth in response to partial hepatectomy were normal after the termination of lactation. Because of the limited scope of this investigation the full implications of these findings are not yet certain.