First Report of Seedling Blight of Eastern Poison Ivy (Toxicodendron radicans) by Colletotrichum fioriniae in Virginia.

Colletotrichum fioriniae is a member of the large cosmopolitan C. acutatum species complex (2). Known agricultural hosts of C. acutatum include apple, European blueberry, grape, olive, papaya, and strawberry (2). In contrast, the life history of C. fioriniae ranges from an epizootic of certain scale insect populations to an endophyte of plants (3,4). The present study extends the phytopathology of C. fioriniae to include poison ivy seedlings. Poison ivy (Toxicodendron radicans) drupes were collected from solitary lianas in Roanoke and Montgomery counties, Virginia. These drupes were subjected to experiments aimed at producing sterile seedlings (1); however, there was extensive blighting and wilting in the germinated seedlings. Associated with the drupes and seedlings was a fungus with white to pale olivaceous grey mycelium with orange blister-like conidiomata and sclerotial masses enclosing the drupe mesocarp as well as conidiomata emerging from blighted, necrotic leaves. Condiomata were plated onto acidified potato dextrose agar (APDA) and oatmeal agar (OA). This consistently yielded colonies identical to those described from diseased tissues and were putatively identified as C. acutatum based on the presence of acervuli containing hyaline, smooth-walled, aseptate conidia with acute ends, the absence of setae, and formation of red pigments in culture (2). Conidial dimensions of four isolates most closely aligned with reported measurements for C. fioriniae (4): mean length ± SD × width ± SD = 15.1 ± 1.7 × 4.9 ± 0.3 µm, L/W ratio = 3.04 on OA. Fungal DNA was isolated and used as template in PCR reactions using oligonucleotide primer pairs corresponding to the internal transcribed spacer (ITS) region, and a portion of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes. The resulting PCR fragments were sequenced and used as queries in BLASTN searches of the GenBank NR database. All of the amplified ITS DNA sequences (497 bp KF944356 and KF944357) were identical to Glomerella/Colletotrichum fioriniae (JN121190 and KF278459). Similarly, the amplified (672 bp) GAPDH sequences (KF944354 and KF944355) were 99.6% similar over the 254 bp overlapping with C. fioriniae (JQ948622). Pathogenicity of two randomly chosen C. fioriniae isolates, TR-123 and TR-126, was confirmed by placing 4.75 mm diam. inoculated agar plugs from 8-day-old fungal cultures or a sterile plug (negative control) at the base of an axenic young seedling ~1.5 to 6.5 cm in height with at least one set of true leaves (1). Each treatment was replicated five times. Acute wilt and blighting of leaves and production of orange acervuli on cotyledons disease symptoms developed by 3 weeks post inoculation (WPI). By 7 WPI all but one of the Colletotrichum-inoculated plants were dead, whereas all of the control plants were healthy with significantly lower area under the disease progress curve values. Colletotrichum was consistently re-isolated, and confirmed morphologically and molecularly, from six of seven diseased seedlings, whereas two of two randomly chosen control seedlings remained asymptomatic and did not yield Colletotrichum. In summary, C. fioriniae may represent a natural biocontrol agent against poison ivy and scale insect herbivores thereof. References: (1) E. Benhase and J. Jelesko. HortScience 48:1, 2013. (2) U. Damm et al. Stud. Mycol. 73:37, 2012. (3) J. Marcelino et al. J. Insect Sci. 9:25, 2009. (4) R. Shivas et al. Fungal Divers. 39:111.

Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana.

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent DeltaRBCS1B::LUC chimeric gene fusion, lacking all 5' transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc(+)) required a homologous recombination event between the DeltaRBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B/ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5' transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc(+) seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc(+) seedlings were isolated, and molecular and genetic analysis indicated that the luc(+) trait was caused by the formation of a recombinant chimeric RBCS3B/1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuffling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossing-over (approximately 3 x 10(-6)), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.

Genetic characterization of a Rhizobium meliloti lactose utilization locus.

We identified several linked genes of a lactose regulon in Rhizobium meliloti. These were lacZ, the structural gene for beta-galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function, lacW and lacX. Insertion mutants in lacW and lacZ belonged to a single genetic complementation group, and lacW appeared to lie upstream of lacZ in an operon. Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose. lacZ was not required for induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual inducer molecule. Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exclusion could not be the sole mechanism. This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.

Rhizobium meliloti mutants with decreased DAHP synthase activity are sensitive to exogenous tryptophan and phenylalanine and form ineffective nodules.

We isolated two Tn5-generated mutants of Rhizobium meliloti whose growth was inhibited by rich medium or by exogenous tryptophan or phenylalanine. These mutants, Rm7479 and Rm7480, belonged to the same genetic complementation group. The mutant locus could not be found on either indigenous megaplasmid but was localized on the chromosome. The mutants formed ineffective nodules on alfalfa plants. They invaded nodules within infection threads and were released into plant cells enclosed within peribacteroid membranes, but once released into the plant cells they failed to differentiate into mature bacteroids. The mutants demonstrated a decrease in total 2-keto-3-deoxy-D-arabino-heptonic acid 7-phosphate synthase (DAHP synthase) activity, which is the first committed step in aromatic biosynthesis. Wild-type genes were isolated that complemented in one case or suppressed in another case, all three mutant phenotypes: growth on rich medium, symbiotic effectiveness, and DAHP synthase activity. Each mutant strain gave rise to linked second-site suppressor mutations that restored growth on rich medium. The suppressor mutants showed restoration of near wild-type DAHP synthase levels. One of the suppressor strains restored effective symbiosis while the other did not. Genetic complementation experiments showed that growth on rich medium, DAHP synthase activity, and effective symbiosis were all affected by the same genetic lesion. These results suggest that normal flux of metabolites through the aromatic biosynthesis pathway is essential for bacteroid development.