86 Wyka et al.: Effects of G. clavigera and L. longiclavatum on Western White Pine for Ophiostomatoid fungi by observing character- istic conidiophores. From this isolation, two species were differentiated based on their morphology, G. clavigera by a more highly branched colony with more elongate and clavate shaped conidia, and L. longiclavatum by a less frequently branching colony with smaller and more cylindrical conidia (Six et al. 2003). Confirmatory identifications by DNA sequencing were conducted by Drs. Wingfield and de Beer. These isolates are now maintained in the culture collection (CMW) of FABI, under G. clavigera #38988 and L. longiclavatum #38989. Fungicide Screening Three systemic injection products including TREE- äge (4% [wt/wt] emamectin benzoate, Syngenta Crop Protection, Greensboro, North Carolina, U.S.), Alamo (14.3% [wt/wt] propiconazole, Syn- genta Crop Protection, Greensboro, North Carolina, U.S.), and Arbotect (20% [wt/wt] thiabendazole, Syngenta Crop Protection, Greensboro, North Carolina, U.S.), were evaluated in vitro to deter- mine their effect on growth of G. clavigera. To do this, serial dilutions of each treatment were estab- lished, from 10,000 to 1 ppm, along with a sterile water control. Individual filter papers, previously sterilized in an autoclave and leſt to dry in a ster- ile environment (1.5 cm, VWR, Radnor, Pennsyl- vania, U.S.), were dipped halfway into respective treatment solutions with sterile forceps and placed flat at the outer edge of an MEA agar plate, with the edge of the paper ~2 cm from the center of the plate (two filter papers per plate, three plates per treatment). Each plate medium was then streaked in the middle with conidia from a one-week old colony of G. clavigera. Researchers did not include L. longiclavatum in the screening because colonies did not survive cold storage. Plates were incubated at 25°C for one week, aſter which time measure- ments were made, and the experiment was con- cluded. The distance from the edge of each filter paper to the closest viable mycelium was measured (repression zone). If contamination obscured the view of the filter paper, no measurement was taken. Mean repression of fungal mycelium was cal- culated in millimeters from six measured repres- sion zones, two per Petri dish, for each treatment. Analysis of variance (ANOVA) for a hierarchical design with nested errors was implemented using ©2016 International Society of Arboriculture the MIXED procedure in SAS (V. 9.3, SAS Institute Inc., Cary, North Carolina, U.S.) with treatment as a fixed effect and dish within treatment as random, followed by comparisons among treatments using Fisher’s protected LSD. A second analysis was per- formed on the subset of treatments obtained by eliminating the control to test for main and interac- tion effects of the factors fungicide and concentra- tion. Demonstration of a highly significant fungicide by concentration interaction on repression was followed by orthogonal linear contrasts (via Esti- mate statements) to test for a response to increased concentration (on a log scale) of each fungicide. SEEDLING EXPERIMENTAL OVERVIEW Following the screening of fungicides in vitro, two experiments with seedlings were conducted. The objectives of the first experiment were to 1) test the pathogenicity of G. clavigera and L. longicla- vatum in WWP seedlings, and 2) test the phyto- toxicity of systemic pesticides in WWP seedlings. The objective of the second experiment was to test whether multiple inoculation points of fungi altered results compared to a single inoculation. Experiment #1 Pathogenicity of fungal isolates Pinus monticola seedlings were purchased from the University of Idaho, Franklin H. Pitkin Forest Nursery (Moscow, Idaho, U.S.), and potted into 15.24-cm pots using a 1:2:1 mix of compost, peat and perlite. From this group of seedlings (average basal diameter = 0.67 cm ± 0.01 SE and average height = 16.8 cm ± 0.5 SE), 50 individuals were selected and randomly assigned to 5 treatments (n = 10 replicates per treatment). The five treatments were 1) G. clavigera inoculation, 2) L. longiclavatum inoculation, 3) co-inoculation of G. clavigera and L. longiclavatum after injection of systemic fun- gicide Alamo, 4) sterile MEA, or 5) de-ionized water. No seedlings were inoculated with a mix of both fungi only. It is known that these fungi are commonly associated and occur in common in nature. The single inoculations were designed to determine if a particular fungal species was more virulent than the other, while co-inoculations, which only followed seedling treatment with Alamo, were used to evaluate fungicidal effects
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