Field efficacy of nonpathogenic Streptomyces species against potato common scab.

AIMS: The primary objective of these experiments was to reduce pathogenicity and virulence of endemic soil pathogenic Streptomyces strains that cause potato common scab (CS) using nonpathogenic Streptomyces strains to suppress CS in a field situation. METHODS AND RESULTS: Nonpathogenic Streptomyces strains that had shown potential for mitigating CS in greenhouse assays were used in Michigan and Pennsylvania fields known to have high CS disease pressure. Five biocontrol (BC) strains and three potato cultivars were used in 2009, and three BC strains and three cultivars were used in 2010 in each location. The effects of BC strains on CS disease incidence and severity differed between locations, years and potato cultivars. When overall means of individual BC treatments were compared with nontreated controls, CS incidence and severity were decreased by all BC strains in PA2009, PA2010 and MI2010, particularly in cultivar 'Yukon Gold' in MI. Biocontrol treatments also significantly shifted the proportions of superficial, raised and pitted lesion types in some cultivar/biocontrol treatment combinations. CONCLUSIONS: All BC strains significantly reduced CS incidence and severity on 'Yukon Gold' in three of four trials, and one BC strain significantly improved the lesion severity profile in cultivar 'Atlantic'. No BC strain significantly reduced CS incidence and severity on all potato cultivars in the different years and locations. SIGNIFICANCE AND IMPACT OF THE STUDY: Several nonpathogenic Streptomyces strains showed potential to reduce CS incidence and severity on two important potato-chipping cultivars in the field. These results can be further applied to reduce CS disease severity in potatoes.

Multiplex real-time PCR (TaqMan) assay for the simultaneous detection and discrimination of potato powdery and common scab diseases and pathogens.

AIMS: To develop a multiplex real-time PCR assay using TaqMan probes for the simultaneous detection and discrimination of potato powdery scab and common scab, two potato tuber diseases with similar symptoms, and the causal pathogens Spongospora subterranea and plant pathogenic Streptomyces spp. METHODS AND RESULTS: Real-time PCR primers and a probe for S. subterranea were designed based on the DNA sequence of the ribosomal RNA ITS2 region. Primers and a probe for pathogenic Streptomyces were designed based on the DNA sequence of the txtAB genes. The two sets of primer pairs and probes were used in a single real-time PCR assay. The multiplex real-time PCR assay was confirmed to be specific for S. subterranea and pathogenic Streptomyces. The assay detected DNA quantities of 100 fg for each of the two pathogens and linear responses and high correlation coefficients between the amount of DNA and C(t) values for each pathogen were achieved. The presence of two sets of primer pairs and probes and of plant extracts did not alter the sensitivity and efficiency of multiplex PCR amplification. Using the PCR assay, we could discriminate between powdery scab and common scab tubers with similar symptoms. Common scab and powdery scab were detected in some tubers with no visible symptoms. Mixed infections of common scab and powdery scab on single tubers were also revealed. CONCLUSIONS: This multiplex real-time PCR assay is a rapid, cost efficient, specific and sensitive tool for the simultaneous detection and discrimination of the two pathogens on infected potato tubers when visual symptoms are inconclusive or not present. SIGNIFICANCE AND IMPACT OF THE STUDY: Accurate and quick identification and discrimination of the cause of scab diseases on potatoes will provide critical information to potato growers and researchers for disease management. This is important because management strategies for common and powdery scab diseases are very different.

First Report of Leveillula taurica Causing Powdery Mildew on Pepper in Maryland.

Pepper plants in large experimental plots in Beltsville, MD developed widespread powdery mildew during the late summer of 2008. Infection was observed in a diversity of accessions that included Capsicum annuum, C. baccatum, C. chinense, and C. frutescens (2). The C. annuum accessions included culinary bell pepper cultivars and breeding lines as well as a diverse collection of ornamental breeding lines, heirlooms, and land races. Significant leaf damage occurred and led to partial defoliation. Extensive coverage of the abaxial surface by white patches of conidia was noted, along with chlorotic regions on the adaxial surface. Conidia were borne singly and were apically tapered, measuring 65.2 ± 3.2 × 14.9 ± 1.9 µm. Cleistothecia were not found on infected leaves (3). PCR amplification of the internal transcribed spacer (ITS) region using ITS1-2 primers yielded a band that was cloned and sequenced (4). The pathogen was identified as Leveillula taurica based on 100% homology to GenBank Accession No. AY912077. Multiple chili pepper and bell pepper plants were inoculated with conidia from an infected bell pepper plant by placement in an enclosed spore deposition chamber for 1 week, with the infected plant suspended over the test plants. Signs of powdery mildew appeared only on inoculated plants. DNA samples from these inoculated plants were analyzed and verified as L. taurica (a sequence was deposited as GenBank No. GQ167201). A second set of inoculations using the newly infected plants confirmed results of the first test, with mildew developing only on inoculated pepper plants. This disease is new to the mid-Atlantic Region of the United States. It has been reported in greenhouse peppers growing in Ontario, Canada where it has become a recurring problem requiring fungicide intervention (1). Given the wide host range of L. taurica and the systemic nature of infections, it is likely that the fungus has become established in Maryland on perennial host plants. References: (1) R. Cerkauskas. Plant Dis. 83:781, 1999. (2) V. de Souza. Plant Pathol. 52:613, 2003. (3) C. Little. Plant Dis. 90:1358, 2006. (4) G. Saenz. Can. J. Bot. 77:150, 1999.

Late Blight Caused by Phytophthora infestans on Solanum sarrachoides in Northeastern Maine.

The area bordering three 110-ha (270-acre) fields of blighted potatoes (Solanum tuberosum L.) in three northeastern Maine locations was surveyed during the summer of 2004 for the occurrence of late blight on cultivated and noncultivated host plants. Special attention was directed to solanaceous weed species. Hundreds of Solanum sarrachoides Sendt. ex. Mart. (hairy nightshade) plants with numerous leaf lesions and moderate defoliation were seen. The frequency of blighted hairy nightshade approximated the frequency of late blight in the adjoining potato fields. Lesions typically contained extensive, white, superficial mycelia colonizing the abaxial and adaxial leaf surfaces. Samples placed in a moist chamber produced lemon-shaped sporangia. On the basis of morphological characteristics, the pathogen was tentatively identified as Phytophthora infestans (Mont.) de Bary. Isolates were obtained by surface-disinfecting leaf sections collected from two locations for 2 to 3 min in 0.5% NaOCl and placing the sections on rye grain medium amended with antibiotics (100 ppm each of penicillin G, pimaricin, and polymyxin). P. infestans was confirmed after reisolating onto rye-lima bean medium. Pathogenicity was tested on detached potato, tomato, and hairy nightshade leaves; the undersides of all leaflets from replicate plants were inoculated with droplets of swimming zoospores (≥500 zoospores per droplet), the leaves were kept at 17°C and 100% humidity, and the extent of sporulation was evaluated after 4, 6, and 7 days. With eight isolates obtained from S. sarrachoides, Koch's postulates were completed on potato and hairy nightshade. Radial growth responses of these strains on rye grain agar amended with 1, 10, or 100 µg per ml of metalaxyl (Ridomil 2E) yielded 50% effective dose values greater than 100 µg per ml, since percentage growth at the highest fungicide concentration exceeded 50% of the no metalaxyl control. These resistance levels are typical of the metalaxyl-insensitive strains of P. infestans isolated from potatoes in this area in recent years, which were previously found to correlate with metalaxyl resistance in bioassays using potato tissues (1). Eight single-sporangial isolates were homozygous for glucose-6-phosphate isomerase and peptidase (Gpi 100/111/122, Pep 100/100). All eight were A2-mating type and mitochondrial haplotype Ia, characteristics common to the US-8 clonal lineage of P. infestans from potato (2), which may infect a wider host range than the old US-1 clonal lineage. When evaluated on differential hosts, three isolates were tomato race PH-1 and complex potato race R 0,1,2,3,4,9,11. DNA fingerprint analysis with probe RG57 further established that the eight hairy nightshade isolates were identical to each other and to local P. infestans isolates from potato. To our knowledge, this is the first report of infection of S. sarrachoides by P. infestans in Maine. The pathogen was previously isolated from this host during field surveys in southern California in the 1980s in connection with late blight of tomato (4). Hairy nightshade has been shown to be a host for US-1, US-8, and US-11 isolates of P. infestans in a laboratory setting (3). The epidemiological significance of S. sarrachoides as an alternative or overwintering host of P. infestans is currently being assessed. References: (1) K. L. Deahl et al. Am. Potato J. 70:779, 1993. (2) S. B. Goodwin et al. Phytopathology 88:939, 1998. (3) H. W. Platt. Can. J. Plant Pathol. 21:301, 1999. (4) V. G. Vartanian and R. M. Endo Plant Dis. 69:516, 1985.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64)and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich pallindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like tmRNA. Our results suggest that mtRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid.

We characterized an unusual tRNA-like sequence that had been found inserted in suppressive variants of the mitochondrial retroplasmid of Neurospora intermedia strain Varkud. We previously identified two forms of the tRNA-like sequence, one of 64 nt (TRL-64) and the other of 78 nt (TRL-78) containing a 14-nt internal insertion in the anticodon stem at a position expected for a nuclear tRNA intron. Here, we show that TRL-78 is encoded in Varkud mitochondrial (mt)DNA within a 7 kb sequence that is not present in Neurospora crassa wild-type 74 A mtDNA. This 7-kb insertion also contains a perfectly duplicated tRNA(Trp)gene, segments of several mitochondrial plasmids and numerous GC-rich palindromic sequences that are repeated elsewhere in the mtDNA. The mtDNA-encoded copy of TRL-78 is transcribed and apparently undergoes 5'- and 3'-end processing and 3' nucleotide addition by tRNA nucleotidyl transferase to yield a discrete tRNA-sized molecule. However, the 14 nt intron-like sequence in TRL-78, which is missing in the TRL-64 form, is not spliced detectably in vivo or in vitro. Our results show that TRL-78 is an unusual tRNA-like species that could be incorporated into suppressive retroplasmids by the same reverse transcription mechanism used to incorporate mt tRNAs. The tRNA-like sequence may have been derived from an intron-containing nuclear tRNA gene or it may serve some function, like mtRNA. Our results suggest that mt tRNAs or tRNA-like species may be integrated into mtDNA via reverse transcription, analogous to SINE elements in animal cells.

Cold-induced freezing tolerance in Arabidopsis.

Changes in the physiology of plant leaves are correlated with enhanced freezing tolerance and include accumulation of compatible solutes, changes in membrane composition and behavior, and altered gene expression. Some of these changes are required for enhanced freezing tolerance, whereas others are merely consequences of low temperature. In this study we demonstrated that a combination of cold and light is required for enhanced freezing tolerance in Arabidopsis leaves, and this combination is associated with the accumulation of soluble sugars and proline. Sugar accumulation was evident within 2 h after a shift to low temperature, which preceded measured changes in freezing tolerance. In contrast, significant freezing tolerance was attained before the accumulation of proline or major changes in the percentage of dry weight were detected. Many mRNAs also rapidly accumulated in response to low temperature. All of the cold-induced mRNAs that we examined accumulated at low temperature even in the absence of light, when there was no enhancement of freezing tolerance. Thus, the accumulation of these mRNAs is insufficient for cold-induced freezing tolerance.

The Arabidopsis thaliana 4-coumarate:CoA ligase (4CL) gene: stress and developmentally regulated expression and nucleotide sequence of its cDNA.

An Arabidopsis cDNA clone encoding 4-coumarate:CoA ligase (4CL), a key enzyme of phenylpropanoid metabolism, was identified and sequenced. The predicted amino acid sequence is similar to those of other cloned 4CL genes. Southern blot analysis indicated that 4CL is single-copy gene in Arabidopsis. Northern blots showed that 4CL expression was activated early during seedling development. The onset of 4CL expression was correlated with the onset of lignin deposition in cotyledons and roots 2-3 days after germination. The timing of the expression of a parsley 4CL1-GUS fusion in transgenic Arabidopsis seedlings was examined in parallel and was very similar to that of endogenous 4CL. In mature plants, highest 4CL expression was observed in bolting stems, where relatively large amounts of lignin accumulate. Both 4CL and 4CL1-GUS mRNA accumulation was strongly and transiently activated by wounding of mature Arabidopsis leaves. 4CL expression was specifically activated within 6 h after infiltration of Arabidopsis ecotype Columbia leaves with a Pseudomonas syringae pv. maculicola strain harboring the bacterial avirulence gene avrB, which causes in incompatible interaction. The timing of 4CL activation was identical to the previously observed activation of PAL gene expression in this interaction. No activation of 4CL expression was observed in a compatible interaction caused by a Pseudomonas syringae pv. maculicola strain without avrB.

The phenylalanine ammonia-lyase gene family in Arabidopsis thaliana.

Phenylpropanoid derivatives are a complex class of secondary metabolites that have many important roles in plants during normal growth and in responses to environmental stress. Phenylalanine ammonialyase (PAL) catalyzes the first step in the biosynthesis of phenylpropanoids, and is usually encoded by a multi-gene family. Genomic clones for three Arabidopsis thaliana PAL genes containing the entire protein-coding region and upstream and downstream sequences have been obtained and completely sequenced. Two A. thaliana PAL genes (PAL1 and PAL2) are structurally similar to PAL genes that have been cloned from other plant species, with a single intron at a conserved position, and a long highly conserved second exon. Previously identified promoter motifs plus several additional sequence motifs were found in the promoter regions of PAL1 and PAL2. Expression of PAL1 and PAL2 is both qualitatively and quantitatively similar in different plant organs and under various inductive conditions. A third A. thaliana PAL gene, PAL3, differs significantly from PAL1 and PAL2 and other sequenced plant PAL genes. PAL3 contains an additional intron, and its deduced amino acid sequence is less homologous to other PAL proteins. The PAL3 promoter region lacks several sequence motifs conserved between A. thaliana PAL1 and PAL2, as well as motifs described in other genes involved in phenylpropanoid metabolism. A. thaliana PAL3 was expressed at very low levels under the conditions examined.

A mitochondrial retroplasmid integrates into mitochondrial DNA by a novel mechanism involving the synthesis of a hybrid cDNA and homologous recombination.

The Mauriceville and Varkud mitochondrial plasmids of Neurospora spp. are closely related, small circular DNAs that propagate via an RNA intermediate and reverse transcription. Although the plasmids ordinarily replicate autonomously, they can also integrate into mitochondrial DNA (mtDNA), yielding defective mtDNAs that in some cases cause senescence. To investigate the integration mechanism, we analyzed four cases in which the Varkud plasmid integrated into the mitochondrial small rRNA gene, three in wild-type subcultures and one in a senescent mutant. Our analysis suggests that the integrations occurred by the plasmid reverse transcriptase template switching between the plasmid transcript and internal sequences in the mitochondrial small rRNA to yield hybrid cDNAs that circularized and recombined homologously with the mtDNA. The integrated plasmid sequences are transcribed, presumably from the mitochondrial small rRNA promoters, resulting in hybrid RNAs containing the 5' segment of the mitochondrial small rRNA linked head-to-tail to the full-length plasmid transcript. Analysis of additional senescent mutants revealed three cases in which the plasmid used the same mechanism to integrate at other locations in the mtDNA. In these cases, circular variant plasmids that had incorporated a mitochondrial tRNA or tRNA-like sequence by template switching integrated by homologous recombination at the site of the corresponding tRNA or tRNA-like sequence in mtDNA. This simple integration mechanism involving template switching to generate a hybrid cDNA that integrates homologously could have been used by primitive retroelements prior to the acquisition of a specialized integration machinery.

Recognition of the avirulence gene avrB from Pseudomonas syringae pv. glycinea by Arabidopsis thaliana.

The response of Arabidopsis thaliana land race Columbia to the bacterial pathogen Pseudomonas syringae pv. maculicola 4326 harboring cloned avirulence genes avrB and avrC from P. syringae pv. glycinea and avrA and avrD from P. syringae pv. tomato was examined. Only avrB was recognized by Columbia, as evidenced by attenuation of disease symptoms, slower bacterial multiplication in planta, and differentially greater induction of mRNA for several defense-related genes. This contrasts with two A. thaliana land races where P. s. pv. maculicola strains containing avrB were not recognized. These plants showed typical disease symptoms, and bacterial multiplication in planta was not reduced in response to P. s. pv. maculicola containing avrB. In addition, there was no differential induction of defense-related mRNAs in these susceptible land races after infiltration with bacteria containing or lacking avrB. These results extend previous observations that avirulence genes from pathogens of one host plant can be recognized by "nonhost" plants and provide the genetic framework for analysis of the plant-specified response to the bacterial avrB gene product in A. thaliana.

Expression dynamics of the tomato rbcS gene family during development.

The tomato rbcS gene family is composed of five genes (rbcS1, 2, 3A, 3B, and 3C) that are differentially expressed during tomato development. Nuclear run-on transcription assays and RNA analysis were used to determine the contribution of transcriptional and post-transcriptional regulation to the accumulation of mRNA from the five rbcS genes in tomato seedlings, leaves, and fruit. We found that the qualitative pattern of mRNA accumulation is regulated at the transcriptional level and that, in general, there is a correlation of rates of rbcS transcription with overall rbcS mRNA abundance in fruit and leaves. Although transcriptional control is a primary determinant for rbcS gene expression in tomato, examination of relative transcription rates and mRNA accumulation of each rbcS gene demonstrated that there is also significant post-transcriptional control of rbcS gene expression during organ development. Individual rbcS mRNAs, which have highly conserved coding sequences and differ only in their 5' and 3' untranslated sequences, have different stabilities. We showed that both transcription and stability of individual rbcS mRNAs are altered in different organs and by the developmental program within these organs as well as by exposure to light. Together, the results provide a comprehensive analysis of the extent of transcriptional and post-transcriptional control that operates within the rbcS gene family during plant development.

Separation of allelic variants by two-dimensional electrophoresis.

The resolving power of two-dimensional (2-D) gel electrophoresis has been tested using 17 allele products at five loci. Standard O'Farrell gels could separate 13 of these isozymes. The addition of a second pH gradient made it possible to separate all but one of the variant proteins. These results indicate that 2-D gel electrophoresis can resolve more than 90% of variants originally detected by one-dimensional (1-D) electrophoresis. The implications of these results for the low estimates of average heterozygosity obtained in surveys using 2-D gel electrophoresis are discussed.