Pedro Alberto Balatti

Molecular cloning and characterization of a sym plasmid locus that regulates cultivar-specific nodulation of soybean by Rhizobium fredii USDA257

Rhizobium fredii strain USDA257 produces nitrogen‐fixing nodules on primitive soybean cultivars such as Peking but fails to nodulate agronomically improved cultivars such as McCall. Transposonmutant 257DH4 has two new phenotypes: it nodulates McCall, and its ability to do so is sensitive to the presence of parental strain U5DA257, i.e. it is subject to competitive nodulation blocking. We have isolated a cosmid containing DNA that corresponds to the site of transposon insertion in 257DH4 and have localized Tn5 on an 8.0 kb EcoRI fragment. The 5596 bp DNA sequence that surrounds the insertion site contains seven open reading frames. Five of these, designated nolBTU, ORF4, and nolV, are closely spaced and of the same polarity. nolWand nolX are of the opposite polarity. The initiation codon for nolW lies 155bp upstream from that of nolB, and it is separated from nolXby 281 bp. The predicted NolT and NolW proteins have putative membrane‐spanning regions. The N‐terminus of the hypothetical NolW protein also has limited homology to NodH of Rhizobium meliloti, but none of the deduced protein sequences has significant homology to known nodulation gene products. Site‐directed mutagenesis with mudll1734 confirms that inactivation of nolB, nolT, nolU, nolV, nolW, or nolX extends host range for nodulation to McCall soybean. This phenotype could not be genetically dissected from sensitivity to competitive nodulation blocking. Expression of nolBTU anti nolX is induced as much as 30‐fold by flavonoid signal molecules, even though these genes lack nod‐box promoters. Histochemical staining of McCall roots inoculated with nolB–, nolU–, or nolX–lacZ fusions verifies that these genes are expressed continuously from preinfection to the stage of the functional nodule. Although a nolU–ORF4–nolV clone hybridizes to a single 8.0 kb EcoRI fragment from 10 strains of R. fredii and broad‐host‐range Rhizobium sp. NGR234, hybridizing sequences are not detectable in other rhizobia.

Transcriptional organization and expression of noIXWBTUV, a locus that regulates cultivar‐specific nodulatlon of soybean by Rhizobium fredii USDA257

Rhizobium fredii is a nitrogen‐fixing bacterial symbiont of soybean and a number of other legume species. We have studied the transcriptional organization of a Sym plasmid locus that restricts the host range of R. fredii USDA257 at both the host species and cultivar level. The genes of this host‐specificity locus, noIXWBTUV, are transcribed from three promoters. Two of these, which are upstream of noIW and noIBTUV, are oriented face to face and initiate transcription at sites that are 14 bp apart. The third lies upstream from noIX. The noIW promoter is constitutive, whereas the noIB and noIX promoters are inducible by flavonoid signals. We have attempted to express genes from this locus in Escherichia coli systems, both in vivo and in vitro. We detected the insert and orientation‐specific expression of two genes, noIX and nolW, but we were unable to obtain expression of noIBTUV. Antiserum raised against NoIT nevertheless detected an abundantly expressed polypeptide of the predicted size in protein extracts of USDA257. This observation, as well as RNA dot blot data from a series of mutants, indicates that noIBTUV is expressed as a single transcriptional unit in R. fredii. Immunological detection of NoIT, and of a second protein, NoIX, was strictly dependent on flavonoid induction. The NoIX protein was larger than the size predicted from the previously published nucleotide sequence, and this led to resequencing and revision of the open reading frame.

Differential regulation of laccase gene expression in Coriolopsis rigida LPSC No. 232.

Two laccase isoenzyme genes (lcc2 and lcc3) from the white-rot fungus Coriolopsis rigida were cloned, and together with the previously described lcc1, their transcript levels were analysed by Quantitative RT-PCR in order to study their expression patterns under a range of putative inducers (Cu(2+), Mn(2+), Fe(3+), 2,6-dimethoxy-1,4-benzoquinone, H(2)O(2,) caffeine, amphotericin B and syringic acid). The highest induction was observed for lcc1 in presence of copper, and thus, a kinetic study was performed to analyze its effect on temporary lcc1 gene expression. Our results showed that upregulation due to copper was linked to growth stage, being highest during the trophophase and decreasing during the idiophase. Amphotericin B increased levels of transcripts of lcc1 and lcc2, syringic acid upregulated lcc1 and lcc3 and 2,6-dimethoxy-1,4-benzoquinone induced lcc2 and lcc3. Possible reasons for why laccase genes from C. rigida are differentially regulated at the transcriptional level are discussed.

Genetic diversity of fast-growing rhizobia that nodulate soybean (Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.

Angular leaf spot: a disease caused by the fungus Phaeoisariopsis griseola (Sacc.) Ferraris on Phaseolus vulgaris L.

Publisher Summary Angular leaf spot (ALS) is a disease of bean ( Phaseolus vulgaris L. ) caused by the fungus Phaeoisariopsis griseola ( Pg ). This chapter discusses the taxonomy, morphological characteristics, and host range of the pathogen. It also describes symptoms of the disease, the disease cycle, and management of the yield loss. Epidemiology, in turn, is the foundation of information-based ALS management. Knowledge of spatial and temporal disease relations and fungal-population biology should have an impact on the disease control. Studies on Pg diversity and epidemiology are integral to characterize and understand the evolution of resistance genes to Pg . Parallel investigations of the molecular determinants of Pg pathogenicity and virulence are critical to understand not only the functions but also the pathways involved in disease resistance. Genetic studies should provide the bases for the development of bean cultivar resistance to Pg . Although the Pg –bean pathosystem is complex, continued basic and applied research should provide answers to solve many of the problems encountered by bean growers.

Genetic diversity among Northwestern Argentinian cultivars of common bean (Phaseolus vulgaris L.) as revealed by RAPD markers

The genetic diversity of 10 commercial cultivars of common beans, developed in Northern Argentina was analyzed based on RAPD markers. Sixteen primers were assayed and among them only 4 showed polymorphisms. A similarity matrix was generated by applying three different association coefficients, Simple Matching, Jaccard and Dice. By the UPGMA method dendrograms were generated and also the principal coordinate analysis was performed. The similarity values found were higher than 40% suggesting that genetic diversity is low. Both cluster analysis and principal coordinates analysis associated commercial cultivars either to the Andean or the Mesoamerican gene pool.

Interaction of Rhizobium fredii USDA257 and nodulation mutants derived from it with the agronomically improved soybean cultivar McCall

Rhizobium fredii USDA257 does not nodulate McCall soybean (Glycine max (L.) Merr.), but two transposon-mutants derived from it, 257DH4 and 257DH5, do. All three organisms cause curling of McCall root hairs and induce the formation of underlying cortical cell divisions. The mutants produce infection threads, and many of the meristematic foci develop into nodules. In contrast, root hairs that deform in response to USDA257 lack infection threads, and meristematic activity ceases prior to the appearance of nodule meristems. Root systems nodulated by mutant 257DH4 reduce acetylene at rates similar to those of roots nodulated by reference R. fredii strain USDA191. The presence of living cells of USDA257 in inocula leads to strong inhibition of nodulation by 257DH4 but not by 257DH5. This blocking effect depends on the ratio of USDA257 cells to 257DH4 cells in the inoculum; nodules that form contain cells of 257DH4, but not those of parental strain USDA257.

Genetic diversity among wild common beans from Northwestern Argentina based on morpho-agronomic and RAPD data

The genetic diversity among 10 wild populations of common bean Phaseolusvulgaris var. aborigineus was analyzed by means of RAPD markers and morpho-agronomic data. The study was performed on populations collected from different sites located in the provinces of Jujuy, Salta and Tucumán in northwestern Argentina. Ten quantitative traits and 33 random primers were scored. Clustering based on morpho-agronomic traits and RAPD markers generated similar phenograms that grouped bean populations based on their site of collection. The levels of diversity observed among populations were low suggesting they have a common ancestor. The levels of diversity shown by morpho-agronomic traits were higher compared to those of molecular markers, most probably due to the effect of the environment. Furthermore, a 480-bp DNA band identified a group of wild populations collected from similar sites. Breeding strategies need to exploit this diversity to broaden the genetic base of commercial beans to develop high yield cultivars.

Characterization of fast-growing rhizobia from nodulated soybean [Glycine max (L.) Merr.] in Vietnam

Summary We have examined six fast-growing Rhizobium strains isolated from nodulated soybean [Glycine max (L.) Merr.] plants growing in Vietnam, with special emphasis on two of them, SMX11 and SMH12. The abilities of strains SMX11 and SMH12 to utilize 11 carbon sources were indistinguishable from that of control Sinorhizobium fredii strains USDA257 and HH103, but their growth rates in standard media were considerably more rapid. The Vietnamese strains also were more salt-tolerant and antibiotic-resistant than the S. fredii reference strains, and their LPS banding patterns were less complex. Weak immunological cross-reactivity between the Vietnamese strains and S. fredii was evident. RFLP patterns of a series of nodulation genes and a repetitive sequence from S. fredii matched those of the Vietnamese strains, as did the sequence of a portion of a 16S rRNA gene. Analysis of cellular fatty acids was consistent with the assignment of strain SMH 12 and four other Vietnamese strains to S. fredii, but it aligned strain SMX11 more closely to other related groups of rhizobia. The Vietnamese strains have broad host ranges for nodulation of legumes, and their abilities to fix nitrogen in association with five soybean cultivars are equivalent to or surpass that of S. fredii.

Pathogenic and molecular variability among isolates of Pyrenophora tritici-repentis , causal agent of tan spot of wheat in Argentina

Tan spot, caused by Pyrenophora tritici-repentis, is a common disease of wheat (Triticum aestivum) responsible for economic losses in some wheat growing areas worldwide. In this study the pathogenic and genetic diversity of 51 P. tritici-repentis isolates collected from different ecological regions of Argentina were analyzed. Virulence tests were conducted on 10 selected wheat cultivars: Buck Halcón, Chris, Gabo, Glenlea, Klein Dragón, Klein Sendero, Max, ND 495, ProInta Guazú and ProInta Imperial. Data revealed significant differences between all main factors evaluated and the interactions for 19 of the isolates analyzed. Based on the reaction type of each isolate/cultivar combination, 48 different pathogenic patterns were detected. The molecular analysis using Inter-Simple Sequence Repeats (ISSR) revealed the existence of 36 different haplotypes among 37 isolates of P. tritici-repentis originally selected for this study. These results indicate that P. tritici-repentis on wheat in Argentina is a heterogeneous fungus, implying that screening wheat germoplasm for resistance for tan spot disease requires a wide range of pathogen isolates.