Joseph M. Vargas

Management of Dollar Spot on Creeping Bentgrass with Metabolites of Pseudomonas aureofaciens (TX-1)

Antifungal extracts from four strains of bacteria that were selected for their ability to inhibit fungal turfgrass pathogens were compared for in vitro activity. The cell extract from Pseudomonas aureofaciens Tx-1 (ATCC 55670) exhibited the greatest antifungal activity against selected turfgrass pathogens. Purification of the extract yielded a single active component that was identified as phenazine-1 carboxylic acid (PCA). Minimum inhibitory concentrations of PCA to tested fungal pathogens ranged from 10 to 25 µg/ml. In greenhouse studies, PCA provided management of dollar spot on creeping bentgrass equal to that of the commercial fungicides triadimefon and chlorothalonil at equivalent rates of active ingredient. Phytotoxic effects were observed on creeping bentgrass in greenhouse but not field evaluations of PCA at the rate of 0.48 g/m2. At the end of 2 years of field study, PCA applied every 14 days at 0.15 g/m2 provided dollar spot management on creeping bentgrass equal to that of chlorothalonil applied every 10 days at the label rate of 0.48 g/m2.

Cloning of a chitinase-like cDNA (hs2), its transfer to creeping bentgrass (Agrostis palustris Huds.) and development of brown patch (Rhizoctonia solani) disease resistant transgenic lines

Abstract We isolated a cDNA clone ( hs2 ) encoding a chitinase-like protein from a triploid Dutch elm disease resistant American elm ( Ulmus americana NPS-3-487). Amino acid sequence of this chitinase-like protein showed 65–78% homology to other plant chitinases. A plasmid KYLX71-pHS2 was constructed using the hs2 sequence under the control of cauliflower mosaic virus 35S promoter and nos terminator. This construct was used to genetically engineer creeping bentgrass ( Agrostis palustris Huds.) via Biolistic® PDS-100/He system. Plasmid JS101 containing the bar herbicide resistance selectable marker gene regulated by the rice actin 1 (Act1) promoter was used as a selectable co-transformation marker. Transgenic plants were produced with a 21.1% transformation frequency on the basis of bialophos selection. The co-transformation frequency for hs2 was 5.68%. Linked transgenes ( bar and mtld ) were co-integrated with a frequency of 100% as expected and 21.3% of the transgenic plants showed integration of all three genes. Southern blot analysis of transformants showed that the hs2 transgene copy numbers ranged from 1 to 5. Northern blot hybridization confirmed transcription of the transgenes, and western blot determined the HS2 protein expression. No correlation was found between gene copy number and the level of gene expression. Disease resistance profiles of five independent transgenic lines in greenhouse trials revealed that two lines (711 and 9603) were significantly ( P Rhizoctonia solani , the causative agent of brown patch disease.

Identification, Characterization, and Distribution of Acidovorax avenae subsp. avenae Associated with Creeping Bentgrass Etiolation and Decline

Bacterial etiolation and decline caused by Acidovorax avenae subsp. avenae is an emerging disease of creeping bentgrass (Agrostis stolonifera) in and around the transition zone, a unique area of turfgrass culture between cool and warm regions of the United States. It is suspected that the disease has been present for many years, although diagnosis of the first occurrence was not reported until 2010. Solicitation of samples from golf courses in 2010 and 2011 was undertaken to investigate the prevalence and dissemination of Acidovorax avenae subsp. avenae on creeping bentgrass. At least 21 isolates from 13 states associated with these outbreaks on golf courses were confirmed as A. avenae subsp. avenae by pathogenicity assays and 16S rDNA sequence analysis at two independent locations. Pathogenicity testing of bacterial isolates from creeping bentgrass samples exhibiting heavy bacterial streaming confirmed A. avenae subsp. avenae as the only bacterium to cause significant disease symptoms and turfgrass decline. Host range inoculations revealed isolates of A. avenae subsp. avenae to be pathogenic on all Agrostis stolonifera cultivars tested, with slight but significant differences in disease severity on particular cultivars. Other turfgrass hosts tested were only mildly susceptible to Acidovorax avenae subsp. avenae infection. This study initiated research on A. avenae subsp. avenae pathogenicity causing a previously uncharacterized disease of creeping bentgrass putting greens in the United States.

First Report of a Bacterial Disease on Creeping Bentgrass (Agrostis stolonifera) Caused by Acidovorax spp. in the United States

In June of 2009, a golf course putting green sample of creeping bentgrass (Agrostis stolonifera L.) cv. Penn G-2 from a golf club in North Carolina was submitted to the Michigan State University Turfgrass Disease Diagnostic Laboratory for diagnosis. The sample exhibited symptoms of general wilt, decline, and characteristic necrosis from the leaf tips down. Fungal pathogens were ruled out when no phytopathogenic fungal structures were observed with microscopic examination of infected tissue. Symptoms appeared similar to those of annual bluegrass affected by bacterial wilt caused by Xanthomonas translucens pv. poae. Bacterial streaming was present in all of the cut infected tissue of the Penn G-2 bentgrass sample when observed with a microscope. To isolate the causal agent, cut leaf tissue (1- to 3-mm tips) exhibiting bacterial streaming was surface disinfected for 1 min in 10% sodium hypochlorite solution and rinsed for 1 min with sterile distilled water. Leaf blades were placed into Eppendorf microtubes with 20 μl of sterile phosphate-buffered saline (PBS) solution (pH 7) and macerated with a sterile scalpel. Serial dilutions up to 1 × 10-4 were performed in sterile PBS; 10 μl of each suspension was plated onto nutrient agar (NA) (Becton Dickinson, Sparks, MD) and incubated at room temperature for 5 days. Pure cultures of three commonly observed single bacterial colonies growing on plates from serial dilutions were made on NA medium. These pure cultures were grown for 5 days and used to inoculate three replicates of 5-week-old Penn G-2 plants that had uniformly filled in 8.5-cm-diameter pots grown under greenhouse conditions. Uninfected Penn G-2 creeping bentgrass plants were inoculated with 1 ml of 1.3 × 109 CFU/ml of bacterial suspension by adding drops of the suspension to blades of sterile scissors used to cut the healthy plants. Of the three different bacterial cultures selected to inoculate healthy plants, only one resulted in slight browning of leaf tips just 2 days after inoculation. The symptoms progressed, and by 5 days after inoculation, browning, twisting and leaf dieback to the sheath were observed. When leaf tips of the inoculated plants were cut, bacterial streaming was observed. Isolation of the bacterium from inoculated Penn G-2 plants was performed to fulfill Kochs postulates. Once isolated, a single bacterial colony was identified by 16S rDNA sequencing (Microcheck Inc. Northfield, VT). 16S rDNA sequencing results indicated that the causal agent of bacterial infection was a member of the Acidovorax genus, with a 100% sequence match to Acidovorax avenae subsp. avenae (2). The same nonflorescent, aerobic, gram-negative bacterium has been consistently isolated from inoculated plants exhibiting symptoms thus far. A member of the Acidovorax genus has also been identified as a pathogen of creeping bentgrass in Japan (1). To our knowledge, this is the first report of a bacterial disease affecting creeping bentgrass caused by Acidovorax spp. in the United States. References: (1) N. Furuya et al. J. Fac. Agric. Kyushu Univ. 54:13. 2009. (2) N. Schaad et al. Syst. Appl. Microbiol. 31:434. 2008.

Recombination of Virulence Genes in Divergent Acidovorax avenae Strains That Infect a Common Host

Bacterial etiolation and decline (BED), caused by Acidovorax avenae, is an emerging disease of creeping bentgrass on golf courses in the United States. We performed the first comprehensive analysis of A. avenae on a nationwide collection of turfgrass- and maize-pathogenic A. avenae. Surprisingly, our results reveal that the turfgrass-pathogenic A. avenae in North America are not only highly divergent but also belong to two distinct phylogroups. Both phylogroups specifically infect turfgrass but are more closely related to maize pathogens than to each other. This suggests that, although the disease is only recently reported, it has likely been infecting turfgrass for a long time. To identify a genetic basis for the host specificity, we searched for genes closely related among turfgrass strains but distantly related to their homologs from maize strains. We found a cluster of 11 such genes generated by three ancient recombination events within the type III secretion system (T3SS) pathogenicity island. Ever since the recombination, the cluster has been conserved by strong purifying selection, hinting at its selective importance. Together our analyses suggest that BED is an ancient disease that may owe its host specificity to a highly conserved cluster of 11 T3SS genes.

Genetic Transformation in Agrostis palustris Huds. (Creeping Bentgrass)

Bentgrass (Agrostis spp.) is the cool-season turfgrass used most commonly on greens in the cool and transitional climatic regions and in the cooler portions of the warm climatic region, especially the arid zone (Beard 1982). The genus Agrostis L. includes a number of species suitable for many turfs, especially in temperate climates with cool, moist summers. The common name bentgrass is applied to all turfgrass species within the genus Agrostis with the exception of redtop. Bentgrasses are generally tolerant of acid soils and can thrive at lower levels of soil fertility than that needed for good performance of Kentucky bluegrass or perennial ryegrass. Some species are annuals but most are perennials and have excellent low-temperature hardiness, including all those utilized for turfgrass purposes.