Usefulness of 10 genomic regions in soybean associated with sudden death syndrome resistance.

Sudden death syndrome (SDS) is an important soybean [Glycine max (L) Merrill] disease caused by the soilborne fungus Fusarium virguliforme. Currently, 14 quantitative trait loci (QTL) had been confirmed associated with resistance or tolerance to SDS. The objective of the study was to evaluate usefulness of 10 of these QTL in controlling disease expression. Six populations were developed providing a total of 321 F2-derived lines for the study. Recombinant inbred lines (RIL) used as parents were obtained from populations of 'Essex' × 'Forrest' (EF), 'Flyer' × 'Hartwig' (FH), and 'Pyramid' × 'Douglas' (PD). Disease resistance was evaluated in the greenhouse at three different planting times, each with four replications, using sorghum infested with F. virguliforme homogeneously mixed in the soil (Luckew et al., Crop Sci 52:2215-2223, 2012). Four disease assessment criteria-foliar disease incidence (DI), foliar leaf scorch disease severity (DS), area under the disease progress curve (AUDPC), and root rot severity-were used. QTL were identified in more than one of the disease assessment criteria, mainly associated with lines in the most resistant categories. Five QTL (qRfs4, qRfs5, qRfs7, qRfs12, and Rfs16) were associated with at least one of the disease assessments across multiple populations. Of the five, qRfs4 was associated with DI, AUDPC, and root rot severity, and Rfs16 with AUDPC and root rot severity. The findings suggest it may be possible for plant breeders to focus on stacking a subset of the previously identified QTL to improve resistance to SDS in soybean.

A New Greenhouse Method to Assay Soybean Resistance to Brown Stem Rot.

Greenhouse, growth chamber, and field experiments were conducted to develop a method to assess resistance of soybeans to Cadophora gregata (Phialophora gregata), causal agent of brown stem rot (BSR). In the new method, C. gregata is introduced at the base of the stems of 2-week-old soybeans, and the presence of the fungus is assessed in the tips of the stems 5 weeks later. To test the effectiveness of the method, two populations of soybeans and 10 checks were inoculated at the stem base and then assayed for fungal colonization of the stem tips, percentage of symptomatic leaflets, and percent internal stem length discolored. The lines also were planted in naturally infested fields to assess for percent internal stem length discolored, and were tested for the presence/absence of a BSR-resistant molecular marker. Greenhouse, field, and molecular marker data were compared. Linear regression analysis suggested that percentage of plants with colonized stem tips explained 41 to 64% of the variability (P < 0.0001) in percent stem length discolored in the field and 58 to 85% of the variability (P < 0.0001) in molecular marker data for BSR resistance. Percent stem length discolored assessed in the greenhouse had the lowest correlation with percent stem length discolored in the field and with the molecular marker. Of three incubation temperatures tested, 22°C was the most conducive for distinguishing resistant/susceptible soybeans using the colonization method.

Soybean phytophthora resistance gene Rps8 maps closely to the Rps3 region.

Root and stem rot is one of the major diseases of soybean. It is caused by the oomycete pathogen Phytophthora sojae. A series of resistance genes (Rps) have been providing soybean with reasonable protection against this pathogen. Among these genes, Rps8, which confers resistance to most P. sojae isolates, recently has been mapped. However, the most closely linked molecular marker was mapped at about 10 cM from Rps8. In this investigation, we attempted to develop a high-density genetic map of the Rps8 region and identify closely linked SSR markers for marker-assisted selection of this invaluable gene. Bulk segregant analysis was conducted for the identification of SSR markers that are tightly linked to Rps8. Polymorphic SSR markers selected from the Rps8 region failed to show cosegregation with Phytophthora resistance. Subsequently, bulk segregant analysis of the whole soybean genome and mapping experiments revealed that the Rps8 gene maps closely to the disease resistance gene-rich Rps3 region.

Resistance to Phialophora gregata Is Expressed in the Stems of Resistant Soybeans.

Growth chamber experiments were conducted to determine if resistance to Phialophora gregata, the causal agent of brown stem rot (BSR) of soybean, is expressed in the stems of resistant soybean genotypes. Upon introduction of the pathogen into the base of stems of 2-week-old seedlings, the fungus advanced with the growing tips of plants of susceptible genotypes but lagged behind in resistant genotypes. Five weeks after introduction of the pathogen, both mean percent stem length colonized by P. gregata and mean percentage of symptomatic trifoliate leaflets were significantly less for resistant than for susceptible genotypes. These results indicate that resistance can be expressed in the stems of resistant soybean plants and suggest that stem inoculation methods may be useful for assessing resistance to P. gregata. Also, in our experiments, internal stem discoloration was not as useful as colonization and foliar symptoms in discriminating resistant from susceptible genotypes.

Genetic tests of the roles of the embryonic ureases of soybean.

We assayed the in vivo activity of the ureases of soybean (Glycine max) embryos by genetically eliminating the abundant embryo-specific urease, the ubiquitous urease, or a background urease. Mutant embryos accumulated urea (250-fold over progenitor) only when lacking all three ureases and only when developed on plants lacking the ubiquitous urease. Thus, embryo urea is generated in maternal tissue where its accumulation is not mitigated by the background urease. However, the background urease can hydrolyze virtually all urea delivered to the developing embryo. Radicles of 2-day-old germinants accumulated urea in the presence or absence of the embryo-specific urease (2 micromoles per gram dry weight radicle). However, mutants lacking the ubiquitous urease exhibited increased accumulation of urea (to 4-5 micromoles urea per gram dry weight radicle). Thus, the ubiquitous and not the embryo-specific urease hydrolyzes urea generated during germination. In the absence of both of these ureases, the background urease activity (4% of ubiquitous urease) may hydrolyze most of the urea generated. A pleiotropic mutant lacking all urease accumulated 34 micromoles urea per gram dry weight radicle (increasing 2.5-fold at 3 days after germination). Urea (20 millimolar) was toxic to in vitro-cultured cotyledons which contained active embryo-specific urease. Cotyledons lacking the embryo-specific urease accumulated more protein when grown with urea than with no nitrogen source. Among cotyledons lacking the embryo-specific urease, fresh weight increases were virtually unchanged whether grown on urea or on no nitrogen and whether in the presence or absence of the ubiquitous urease. However, elimination of the ubiquitous urease reduced protein deposition on urea-N, and elimination of both the ubiquitous and background ureases further reduced urea-derived protein. The evidence is consistent with the lack of a role in urea hydrolysis for the embryo-specific urease in developing embryos or germinating seeds. Because the embryo-specific urease is deleterious to cotyledons cultured in vitro on urea-N, its role may be to hydrolyze urea in wounded or infected embryos, creating a hostile environment for pest or pathogen. While the ubiquitous urease is operative in leaves and in seedlings, all or most of its function can be assumed by the background urease in embryos and in seedlings.

Pleiotropic soybean mutants defective in both urease isozymes.

Two new soybean [Glycine max (L.) Merr. cv. Williams] loci, designated Eu2 and Eu3, were identified in which ethyl methanesulfonate (EMS)-induced mutation eliminated urease activity. These loci showed no linkage to each other or to the "Sun-Eul" locus described in the accompanying paper (Meyer-Bothling and Polacco 1987). Unlike sun (seed urease-null) mutations those at Eu2 and Eu3 affected both urease isozymes: the embryo-specific (seed) and the ubiquitous (leaf) urease. The eu2/eu2 mutant had no leaf activity and 0.6% normal seed activity. Two mutant Eu3 alleles were recovered, eu3-e1 and Eu3-e3. The eu3-e1/eu3-e1 genotype lacked both activities while Eu3-e3/Eu3-e3 had coordinately reduced leaf (0.1%) and seed (0.1%) activities. Only the Eu3-e3 mutation showed partial dominance, yielding about 5%-10% normal activity for each urease in the heterozygous state. Each homozygous mutant contained normal levels of embryo-specific urease mRNA and protein subunit, both of normal size. However, urease polymerization was aberrant in all three mutants. In all cases where urease could be measured, it was found to be temperature sensitive and, in addition, the embryo-specific urease of Eu3-e3/Eu3-e3 had an altered pH dependence. These mutants may be defective in a urease maturation function common to both isozymes as suggested by the normal levels of urease gene product, coordinately (or nearly so) reduced urease isozyme activities, temperature sensitivity in both ureases (Eu3-e3) and the non-linkage of Eu2 and Eu3 to the locus encoding embryo-specific urease (Sun-Eul). Ubiquitous urease activity is reduced in mutant seed coat and callus culture as well as in leaf and cotyledon tissue. No mutant callus utilized urea (5 to 10 nM0 as sole nitrogen source. However, all mutant cell lines tolerated normally toxic levels of urea (25 to 250 mM) added to medium containing KNO3/NH4No3 as nitrogen source. Urea thus may be used in cell culture as a selection agent for phenotypes either lacking or regaining an active ubiquitous urease.

Conditional lethality involving nuclear and cytoplasmic chlorophyll mutants in soybeans.

A conditionally lethal phenotype occurred when a nuclear chlorophyll mutant (y 20-k 2) was present with a cytoplasmic chlorophyll mutant (cyt-Y 2) in soybean (Glycine max [L.] Merr.). Nuclear mutant y 20-k 2, Genetic Type Collection Number T253, has yellow foliage, tan-saddle-pattern seed and is viable. The y 20-k 2 mutant cannot be separated by classical genetic tests into two separate components, y 20 (yellow foliage) and k 2 (tan-saddle-pattern seed). Mutant cyt-Y 2, T275, is inherited cytoplasmically, has yellow foliage, and is viable. The genotype cyt-Y 2 y 20-k 2/ y 20-k 2 is a conditional lethal; the genotype is lethal under field conditions, but plants survive under greenhouse conditions. This interaction is unique to y 20-k 2. This conditionally lethal genotype may be useful in molecular studies on the interaction between nuclear and plastid genomes.