Unlocking a Mystery: Characterizing the First Appearance of Clavibacter nebraskensis in Mexican Cornfields.

The Goss's wilt and leaf blight is a disease of maize (Zea mays) caused by Clavibacter nebraskensis, which was widespread in the last several years throughout the Midwest in the United States, south in Texas, and north to Canada. The bacterium is included within the high-risk list of quarantine pathogens by many plant protection organizations and countries including Mexico. Severe blight symptoms on maize plants were found in different provinces from Coahuila and Tlaxcala, Mexico, in 2012 and 2021, respectively. Twenty bacterial isolates with morphology similar to C. nebraskensis were obtained from the diseased maize leaves. The isolates were confirmed by phenotypic tests and 16S rRNA and gyrB sequencing. Two strains were tested for pathogenicity tests on seven hybrid sweet corn cultivars available in Mexico, and the most sensitive cultivar was tested for all the strains to fulfill Koch's postulates. The phylogenetic reconstruction based on two single loci reveals a remarkable clustering of Mexican strains to American strains reported approximately 50 years ago. The presence of this pathogen represents a risk and a significant challenge for plant protection strategies in Mexico and maize diversity.

Reclassification of subspecies of Acidovorax avenae as A. Avenae (Manns 1905) emend., A. cattleyae (Pavarino, 1911) comb. nov., A. citrulli Schaad et al., 1978) comb. nov., and proposal of A. oryzae sp. nov.

The bacterium Acidovorax avenae causes disease in a wide range of economically important monocotyledonous and dicotyledonous plants, including corn, rice, watermelon, anthurium, and orchids. Genotypic and phenotypic relatedness among strains of phytopathogenic A. avenae subsp. avenae, A. avenae subsp. citrulli, A. avenae subsp. cattleyae and A. konjaci, as well as all other Acidovorax species, including A. facilis, the type strain of Acidovorax, was determined. The 16s rDNA sequencing confirmed previous studies showing the environmental species to be very distant from the phytopathogenic species. DNA/DNA reassociation assays on the different strains of A. avenae revealed four (A, B, C, and D) distinct genotypes. Taxon A included six A. avenae subsp. avenae strains from corn that had a mean reciprocal similarity of 81%; taxon B included six A. avenae subsp. avenae strains from rice that had a mean reciprocal similarity of 97%; taxon C contained 11 A. avenae subsp. citrulli strains from cucurbits (cantaloupe, watermelon, and pumpkin) that had a mean reciprocal similarity of 88%, and taxon D contained four A. avenae subsp. cattleyae strains from orchids that had a mean similarity of 98%. The mean reciprocal relatedness between taxa A, B, C, and D was less than 70%. Sequence analysis of 16S rDNA and the 16S-23S rDNA internally transcribed spacer region, as well as AFLP analysis, revealed the same four taxa. All four were easily differentiated phenotypically from each other and from all other recognized Acidovorax species. Strains of A. avenae did not contain 3-hydroxyoctanoic acid, which was found in all other species. On the basis of these and previous genetic and phenotypic results, we propose an emendation of the species A. avenae. A. avenae subsp. citrulli (C strains) and A. avenae subsp. cattleyae (D strains) should be elevated to species rank as A. citrulli and A. cattleyae, respectively. We further propose a new taxon for the B strains, A. oryzae sp. nov. with FC-143T = ICPB 30003T = ICMP 3960T = ATCC 19882T as the type strain.

Plasmid transformation and expression of the firefly luciferase in Microbacterium testaceum type and endophytic colonizing field strains.

Microbacterium testaceum is a predominant endophytic bacterial species isolated from corn and sorghum in the midwestern United States. The development of genetic transfer systems for M. testaceum may enable its use for biocontrol and other applications. The type strain (IFO 12675) and field isolates (SE017, SE034, and CE648) were grown to mid-exponential phase, concentrated (1.0 x 1011 CFU x mL(-1)), electroporated (Escherichia coli-Clavibacter shuttle plasmid pDM302), and plated on TSA with 10 microg x mL(-1) chloramphenicol. Transformation efficiencies averaged 140 CFU x microg(-1) of DNA. Restriction endonuclease analysis showed that pDM302 was not altered after extraction from transformants and re-introduction into E. coli. Transformants with pDM302 were also subjected to nonselective growth conditions, with the frequency of loss after one passage being 84% for IFO 12675 and 88% for SE034. We inserted the green fluorescent protein and the firefly luciferase (FFlux) reporter genes into pDM302, confirming the expression of FFlux in IFO 12675 and SE034. The SE034 FFlux strain was recovered from inoculated corn in greenhouse studies and found to fluoresce by luminometry. These results in M. testaceum demonstrate for the first time its transformability, pDM302 replication, FFlux gene expression, and the recovery of the FFlux recombinant strain from inoculated corn.

Divergence and mosaicism among virulent soil phages of the Burkholderia cepacia complex.

We have determined the genomic sequences of four virulent myophages, Bcep1, Bcep43, BcepB1A, and Bcep781, whose hosts are soil isolates of the Burkholderia cepacia complex. Despite temporal and spatial separations between initial isolations, three of the phages (Bcep1, Bcep43, and Bcep781, designated the Bcep781 group) exhibit 87% to 99% sequence identity to one another and most coding region differences are due to synonymous nucleotide substitutions, a hallmark of neutral genetic drift. Phage BcepB1A has a very different genome organization but is clearly a mosaic with respect to many of the genes of the Bcep781 group, as is a defective prophage element in Photorhabdus luminescens. Functions were assigned to 27 out of 71 predicted genes of Bcep1 despite extreme sequence divergence. Using a lambda repressor fusion technique, 10 Bcep781-encoded proteins were identified for their ability to support homotypic interactions. While head and tail morphogenesis genes have retained canonical gene order despite extreme sequence divergence, genes involved in DNA metabolism and host lysis are not organized as in other phages. This unusual genome arrangement may contribute to the ability of the Bcep781-like phages to maintain a unified genomic type. However, the Bcep781 group phages can also engage in lateral gene transfer events with otherwise unrelated phages, a process that contributes to the broader-scale genomic mosaicism prevalent among the tailed phages.

Reclassification of Xanthomonas campestris pv. citri (ex Hasse 1915) Dye 1978 forms A, B/C/D, and E as X. smithii subsp. citri (ex Hasse) sp. nov. nom. rev. comb. nov., X. fuscans subsp. aurantifolii (ex Gabriel 1989) sp. nov. nom. rev. comb. nov., and X. alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 sp. nov. nom. rev. comb. nov.; X. campestris pv malvacearum (ex smith 1901) Dye 1978 as X. smithii subsp. smithii nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones, 1935) dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nom. rev.; and "var. fuscans" of X. campestris pv. phaseoli (ex Smith, 1987) Dye 1978 as X. fuscans subsp. fuscans sp. nov.

Bacterial canker of citrus is a serious disease of citrus worldwide. Five forms of the disease have been described, cankers "A", "B", "C", "D", and "E". Although considerable genetic diversity has been described among the causal agents of the five forms of citrus canker and supports multiple taxons, the causal agents currently are classified as pathovars citri ("A"), aurantifolii ("B/C/D") and citrumelo ("E") of a single species, Xanthomonas campestris pv. citri (or X. axonopodis pv. citri). To determine the taxonomic relatedness among strains of X. campestris pv. citri, we conducted DNA-DNA relatedness assays, sequenced the 16S-23S intergenic spacer (ITS) regions, and performed amplified fragment length polymorphism (AFLP) analysis, using 44 strains representative of the five recognized forms of citrus canker. Under stringent DNA reassociation conditions (Tm - 15 degrees C), three distinct genotypes of citrus pathogens were revealed: taxon I included all "A" strains; taxon II contained all "B", "C", and "D" strains; and taxon III contained all "E" strains. The three citrus taxa showed less than 50% (mean) DNA-DNA relatedness to each other and less than 30% (mean) to X. campestris pv. campestris and X. axonopodis pv. axonopodis. Taxa I and II strains share over 70% DNA relatedness to X. campestris pv. malvacearum and X. campestris pv. phaseoli var. fuscans, respectively (at Tm - 15 degrees C). Taxon III strains share 70% relatedness to X. campestris pv. alfalfae. Previous and present phenotypic data support these DNA reassociation data. Taxon II strains grow more slowly on agar media than taxa I and III strains. Taxa I and III strains utilize maltose, and liquefy gelatin whereas taxon II strains do not. Taxon I strains hydrolyze pectate (pH 7.0) whereas Taxon II strains do not. Taxon III strains utilize raffinose whereas Taxon I strains do not. Each taxon can be differentiated by serology and pathogenicity. We propose taxa I, II, and III citrus strains be named, respectively, Xanthomonas smithii subsp. citri (ex Hasse, 1915) sp. nov. nom. rev. comb. nov., Xanthomonas fuscans subsp. aurantifolii (ex Gabriel et al., 1989) sp. nov. nom. rev. comb. nov., and Xanthomonas alfalfae subsp. citrumelo (ex Riker and Jones) Gabriel et al., 1989 nov. rev. comb. nov. Furthermore, based on the analysis of 40 strains of 19 other xanthomonads, we propose to reclassify X. campestris pv. malvacearum (ex Smith, 1901) Dye 1978 as X. smithii subsp. smithii sp. nov. comb. nov. nom. nov.; X. campestris pv. alfalfae (ex Riker and Jones) Dye 1978 as X. alfalfae subsp. alfalfae (ex Riker et al., 1935) sp. nov. nov. rev.; and "var. fuscans" (ex Burkholder 1930) of X. campestris pv. phaseoli (ex Smith, 1897) as X. fuscans subsp. fuscans sp. nov.

The accidental plant pathologist.

This article presents the experiences of a woman in academic plant pathology from the 1950s to today. Topics include the social climate for women in science, personal and professional developments and research discoveries, public policy issues in agriculture and biotechnology affecting plant pathology, and projections for the future of plant pathology.

An evolutionary perspective of Pierce's disease of grapevine, citrus variegated chlorosis, and mulberry leaf scorch diseases.

Xylella fastidiosa causes diseases on a growing list of economically important plants. An understanding of how xylellae diseases originated and evolved is important for disease prevention and management. In this study, we evaluated the phylogenetic relationships of X. fastidiosa strains from citrus, grapevine, and mulberry through the analyses of random amplified polymorphic DNAs (RAPDs) and conserved 16S rDNA genes. RAPD analysis emphasized the vigorous genome-wide divergence of X. fastidiosa and detected three clonal groups of strains that cause Pierce's disease (PD) of grapevine, citrus variegated chlorosis (CVC), and mulberry leaf scorch (MLS). Analysis of 16S rDNA sequences also identified the PD and CVC groups, but with a less stable evolutionary tree. MLS strains were included in the PD group by the 16S rDNA analysis. The Asiatic origins of the major commercial grape and citrus cultivars suggest the recent evolution of both PD and CVC disease in North and South America, respectively, since X. fastidiosa is a New World organism. In order to prevent the development of new diseases caused by X. fastidiosa, it is important to understand the diversity of X. fastidiosa strains, how strains of X. fastidiosa select their hosts, and their ecological roles in the native vegetation.

Uses of antimicrobials in plant agriculture.

Bacterial diseases of plants are less prevalent than diseases caused by fungi and viruses. Antimicrobials for prophylactic treatment of bacterial diseases of plants are limited in availability, use, and efficacy, and therapeutic use is largely ineffective. Most applications are by spray treatments in orchards. Monitoring and surveillance for drug resistance are not routinely done. In the United States, data on use of antimicrobials for treatment of bacterial diseases of plants are limited to streptomycin and oxytetracycline. Resistance to streptomycin has become widespread among bacterial phytopathogens; no resistance among these bacteria has yet been reported for oxytetracycline. No human health effects have been documented since inception of use of antimicrobials in plants in the 1950s. Transfer of antimicrobial resistance from marker genes in transgenic plants to bacteria has not been documented under natural conditions in field-grown plants. However, antimicrobial-resistance genes are being eliminated from use as marker genes because of concerns about possible transfer from plant genomes back to bacteria, with further horizontal transfer to the bacteria in the environment, or from plant genomes to animals by plant consumption. No new antimicrobials are expected to be used in plant agriculture because of high costs of development, regulatory constraints, and environmental and human health concerns. Alternatives to antimicrobials, such as biocontrol agents, transgenic plants, and novel chemicals, are being developed and marketed, although their efficacy remains to be determined.

Isolation and characterization of endophytic colonizing bacteria from agronomic crops and prairie plants.

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log(10) CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log(10) CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log(10) CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log(10) CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.