June 20-23, 2010 - Vancouver, British Columbia Canada
(Joint with the Canadian Phytopathological Society)
Potential for forecasting late blight on specialty potatoes in western Washington of the U.S.G. Babette (1), D. A. INGLIS (1)
(1) Washington State University Mount Vernon NWREC
This study focused on adapting a disease forecasting system called WISDOM (University of Wisconsin) for predicting late blight epidemics in specialty potato production systems of western Washington (WWA). Although disease forecasting is generally known to reduce unnecessary fungicide sprays and application costs in crop production, none have been developed with potato late blight, potato seed piece inoculum, and WWA deliberately in mind. Using 12 years of historical environmental and late blight data accumulated at WSU-NWREC in Mount Vernon, WISDOM correctly predicted late blight lesion onset 58% (7 out of 12 years) and late blight lesion spread 92% (11 out of 12 years) of the time. WISDOM-predicted fungicide spray intervals ranged between 5 and 10 days. During low disease pressure years (1996, 2001, 2003, 2009) WISDOM advised 16 fewer sprays while during medium-to-high disease pressure years (1994, 1995, 1997, 1999, 2000, 2002, 2008) WISDOM advised applying 3 more sprays than used in WSU potato research plots. Savings in fungicide costs and quantities in WWA will most likely be realized from eliminating unnecessary late blight sprays in a low disease pressure year rather than in a medium or high disease pressure year. However to use late blight forecasting successfully in WWA, it must be integrated with (i) routine late blight seed piece fungicide treatment, (ii) a calendar foliar fungicide spray at crop green-row stage, and (iii) comprehensive late blight sanitation practices.
Quantitative and qualitative variations in expression of pathogenicity genes of Pythium aphanidermatum during root rot infection of different hosts
K. BALA (4), G. P. Robideau (1), T. L. Rintoul (1), N. Tisserat (2), J. P. Hamilton (3), R. C. Buell (3), A. C. Lévesque (1), B. Saville (5)
(1) Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, CANADA; (2) Dept. of Horticulture and Landscape Architecture, Colorado State University, Fort Collins, CO, U.S.A.; (3) Dept. of Plant Biology, Michigan State University, East Lansing, MI, U.S.A.; (4) Forensic Science Program, Trent University, Peterborough & Agriculture and Agri-Food Canada, Ottawa, ON K1A 0C6, CANADA; (5) Forensic Science and Environmental and Life Sciences Graduate Program, Trent University, Peterborough, ON, K9J 7B8, CANADA
Pythium aphanidermatum is a major pathogen of greenhouse vegetables and several other economically important crops. To investigate differences in gene expression during infection of different hosts, we are planning to analyze the transcriptome of P. aphanidermatum at different time points during disease development in seedlings of monocot and dicot crops. The annotated genome of P. ultimum var. ultimum DAOM BR144 (43Mb) and the assembled but unannotated genome of P. aphanidermatum have been used to first investigate the putative secretome of P. aphanidermatum. Similar to P. ultimum, P. aphanidermatum does not seem to have the classical RXLR effector proteins. However, it does contain necrosis inducing proteins, serine and cysteine protease inhibitors, various proteases, as well as CBEL-like and ras-like proteins. Reverse transcriptase assays will be developed for some of the genes coding these proteins to establish the optimum treatments and timings for extraction of RNA to study host pathogen interaction differentials. Gene expression profiles under different host crops during root rot disease development will be investigated with next generation sequencing technology. Data from these studies will be also used to annotate the genome of P. aphanidermatum.
Cross-pathogenicity of Verticillium dahliae isolates from skullcap and peppermint
J. K. DUNG (2), L. J. du Toit (1), E. W. Gatch (1), D. A. Johnson (2)
(1) Washington State University Mount Vernon NWREC, Mount Vernon, WA, U.S.A.; (2) Washington State University, Pullman, WA, U.S.A.
Skullcap (Scutellaria lateriflora, Lamiaceae) is a herb used in alternative medicine. A commercial skullcap crop with foci of wilted and necrotic plants was examined in Washington in 2008. Three fungal isolates from plants were identified as Verticillium dahliae (Vd) based on morphology and a Vd-specific PCR assay. Skullcap and peppermint (Mentha x piperita) cultivars ‘Black Mitcham’ (susceptible to Verticillium wilt) and ‘Redefined Murray’ (moderately resistant) were inoculated with a peppermint, a potato, and three skullcap isolates using a soil drench (105 conidia/cm3). Disease ratings were converted to area under disease progress curves (AUDPC), and aboveground biomass was recorded. All skullcap isolates caused typical Verticillium wilt symptoms (chlorosis, wilting and necrosis) on skullcap and ‘Black Mitcham’. Isolates from skullcap and peppermint caused significantly greater (P < 0.05) AUDPC values and reduced yields on skullcap and ‘Black Mitcham’ compared to the potato isolate. One skullcap isolate caused significantly greater AUDPC values on ‘Redefined Murray’ than the other isolates. The potato isolate caused mild or no symptoms on all hosts. V. dahliae was observed on 5% of seeds harvested from skullcap plants inoculated with the mint isolate, but 0% of seeds from plants inoculated with the other isolates. This is the first report of Vd infecting skullcap, cross-pathogenicity among isolates from skullcap and peppermint, and seedborne Vd in skullcap.
A community-based stream monitoring program in western Washington for early detection of invasive Phytophthora spp.
M. ELLIOTT (1), G. Chastagner (1), K. P. Coats (1), A. DeBauw (1), K. Riley (1)
(1) Puyallup Research and Extension Center, Washington State University, Puyallup, WA, U.S.A.
To supplement state agencies in their monitoring for Phytophthora ramorum, the sudden oak death (SOD) pathogen, a community-based stream monitoring program was begun in 2010. This project will expand on the streams currently being sampled by the WA Dept. of Natural Resources (WADNR) as part of the national P. ramorum survey and on nursery surveys by WA State Department of Agriculture (WSDA) and will allow for early detection of P. ramorum and other invasive Phytophthora species, as well as examining the biodiversity of Phytophthora spp. in stream ecosystems. Sites were chosen based on input from WSDA and WADNR and on volunteer availability. The baiting process involves placing Rhododendron ‘Nova Zembla’ leaves in mesh bags and deploying them in the stream for two weeks. Four sites are being monitored for six intervals and three sites for one two-week baiting period. After bait retrieval the leaves are cultured on Phytophthora-selective media and colonies of Phytophthora are isolated onto V8 agar. Phytophthora species are identified using molecular and cultural methods. Volunteers consist of Master Gardeners, high school and college students, and others. In addition to baiting, some of the student groups are doing research projects on Phytophthora in the lab as part of their class requirements. More baiting sites are planned for 2011.
Further studies on zoospore germination inhibition of three lineages of Phytophthora ramorum by chemical fungicides, and identification of a potential bacterial biocontrol agent
M. ELLIOTT (3), S. F. Shamoun (2), G. Sumampong (2), D. James (1), S. Masri (1), A. Varga (1)
(1) Canadian Food Inspection Agency, Sidney Laboratory, Sidney, BC, CANADA; (2) Canadian Forest Service, Pacific Forestry Centre, Victoria, BC, CANADA; (3) Puyallup Research and Extension Center, Washington State University, Puyallup, WA, U.S.A.
Thirteen fungicides with varying modes of action were evaluated for their effectiveness on inhibiting zoospore germination of Phytophthora ramorum. Nine isolates of P. ramorum representing the three clonal lineages were tested. During this study some of the isolates were found to have bacteria associated with the zoospores. We have observed that when bacterial contamination is present, very little or no sporangia are produced. Three morphologically different bacteria have been observed under the compound microscope from contaminated cultures. It is our intention to purify these three bacteria and identify them using the 16s rDNA sequence. The biological significance of these bacteria and their potential use as biocontrol agents will be explored. All of the fungicides were effective in preventing zoospore germination at the recommended doses for controlling Phytophthora disease on ornamental plants. EC50 values were lower for P. ramorum on many of the fungicides than for other Phytophthora spp. commonly found in agriculture, suggesting that resistance has not yet developed in this species. There was variability among isolates in their sensitivity to different chemicals. Use of some chemical fungicides and biological control agents can be an effective tool in managing P. ramorum in nurseries as part of an integrated pest management program.
Fungal species associated with coast live oak (Quercus agrifolia) mortality in Southern California
A. ESKALEN (3), S. C. Lynch (1), P. Zambino (4), T. Scott (2)
(1) Center for Conservation Biology, University of California, Riverside; (2) Department of Earth Science, University of California, Riverside; (3) Department of Plant Pathology and Microbiology, University of California, Riverside; (4) USDA Forest Service, Pacific Southwest Region, San Bernardino, CA
Sharp decline and mortality of coast live oak (Quercus agrifolia) has been observed in San Diego County, California, since 2002. Much of this decline has been attributed to a new pest in California, the goldspotted oak borer (GSOB, Agrilus coxalis). Associated symptoms of crown thinning, bark cracking and/or peeling, patches of stain (1–10 cm diameter) and bleeding on the bole, and tree death have mostly been observed on individuals over 30 cm diameter at breast height (DBH). In 2008, a Botryosphaeria species was recovered from necrotic tissue of bleeding bole cankers from GSOB-affected trees in Jamul, San Diego County. Zone lines separated dead and live tissue in affected phloem and xylem. A survey of oak stands throughout San Diego and Riverside Counties in 2009–2010 consistently recovered three Botryosphaeria species along with Biscogniauxia mediterranea, Togninia fraxinopennsylvanica, Hypoxylon sp. Daldinia sp. Pezicula sp., Bionectria sp., Phialophora sp., Diatrypella sp., Fusarium sp. from the bark of bleeding trunk and branch cankers at all eight locations—those both within and extending to 20 miles beyond areas infested by GSOB, where tree mortality has been observed. Species identification was confirmed by ITS4/5 rDNA sequence comparisons in Genbank. Pathogenicity tests are underway.
Development of a soil bioassay to assess the risk of spinach Fusarium wilt
E. W. GATCH (1), L. J. du Toit (1)
(1) Washington State University NWREC Mount Vernon, WA, U.S.A.
Mild winters, dry summers, and high latitude make the maritime Pacific Northwest U.S.A. uniquely suited to production of spinach seed. However, the acid soils of this region are highly conducive to spinach Fusarium wilt (Fusarium oxysporum f. sp. spinaciae). Despite field applications of limestone and crop rotations of up to 15 years to suppress the disease, severe losses still occur. There is currently no selective medium or molecular assay for quantifying the pathogen in soil. The objective of this study was to develop a greenhouse soil bioassay to assess the relative risk of spinach Fusarium wilt for individual fields. Soils were sampled from three fields shown to differ in inoculum potential based on preliminary evaluation. Subsamples of each soil were pasteurized at three levels to obtain a range of inoculum densities. Three female spinach lines (highly susceptible, moderately susceptible, and moderately resistant to Fusarium wilt) were planted in each soil-pasteurization combination and rated weekly for Fusarium wilt severity. Soil, spinach line, and pasteurization treatments significantly affected disease severity and plant biomass. An index of wilt severity effectively differentiated the disease potential associated with each treatment. To test the bioassay further, growers submitted soil sampled from 26 fields intended for spinach seed production in 2010. Each soil and the appropriate soil control treatments were assayed using the same three spinach lines. Soil, spinach line, and the interaction between these factors significantly affected severity of Fusarium wilt, with disease ratings 28 and 35 days after planting providing optimum differentiation of Fusarium wilt risk among the soils. The accuracy of these wilt predictions will be determined by evaluating Fusarium wilt severity in spinach seed crops grown in these fields in 2010.
Development of a quantitative real-time PCR assay for assessment of Phytophthora rubi in soil
J. A. GIGOT (1), T. Walters (1)
(1) Washington State University-NWREC Mount Vernon, WA, U.S.A.
Phytophthora rubi is a serious pathogen of raspberry in northwestern Washington. A real-time PCR assay was developed to quantify P. rubi inoculum in soil using primers and a TaqMan™ probe adapted from a previous publication. A standard curve was created for the TaqMan assay using P. rubi DNA amounts ranging from 1 ng to 1 fg. In order to relate DNA quantity from the standard curve to propagule density in soil, DNA was extracted from triplicate samples of sterile field soil infested with P. rubi oospores at densities of 0, 10, 100 and 1000 oospores/g soil. To assess the effect of these oospore densities of P. rubi on raspberry root rot, a greenhouse bioassay was developed. Raspberry tissue culture plants (cv. Meeker) were planted into sterile field soil infested with oospores (0, 10, 100 or 1000/g soil) and grown in conetainers for ~6 weeks (6 replications, 2 tests). Plants were analyzed for root rot disease on a 0 to 9 scale (0 = healthy, 9 = severe symptoms). Mean cycle threshold values for DNA extracted from infested field soil was significantly (P < 0.05) different between 1000 (28.8) versus 10 (31.2) and 0 (35.7) oospores/g soil. In the greenhouse bioassay, there were significant differences in root rot ratings among raspberry plants inoculated with 1000 (7.7), 10 (3.3) and 0 (0.5) oospores per gram of soil. Further work is needed to optimize DNA extraction efficiency from soil. This real-time assay will be useful in future studies on the impact of soil treatments with chemical fumigants or alternative practices on the fate of P. rubi inoculum in soil.
Purification and characterization of novel glucanases from Trichoderma harzianum ETS 323
S. LIU (1), H. Jhan (2), C. Chen (1), K. Peng (2)
(1) Department of Molecular Biotechnology, Da-Yeh University, Changhua, TAIWAN; (2) Institute of Biotechnology, National Dong-Hwa University, Hualien, TAIWAN
Trichoderma harzianum ETS 323 secretes at least two glucanases, a 23.5 kDa endoglucanase (EG Th1) and a 61 kDa exoglucanase (ExG Th1) that have important roles in biocontrol mechanism. Both the enzymes were identified by their reaction products and were purified to homogeneity. The temperature and pH optima for EG Th1 (7.3 fold purification, yield 5.0%) and ExG Th1 (33.7 fold purification, yield 0.15%) were 50°C and pH 4.5, respectively. Kinetic parameters of EG Th1 (Km: 23 mg mL–1, Vmax: 294 μM min–1, specific activity: 7.4 U mg–1) and ExG Th1 (Km: 85 mg mL–1, Vmax: 385 μM min–1, specific activity: 24.6 U mg–1) towards carboxymethyl cellulose (CMC) were determined. Both enzymes favored CMC and MnCl2 over other substrates tested and maintained 100% activity for 10 days at 38°C. Metal ions such as KCl, MgCl2, HgCl2, and FeCl3 showed approximately 30% inhibition against EG Th1 but not ExG Th1. Both enzymes catalyzed transglycosylation of glucose in the presence of cellobiose; however, ExG Th1 exhibited better activity and higher product diversity.
Identification of expressed sequences in lily under pathogen attack and rhizobacterium induction
Y. Liu (1), K. Yang (1), C. CHEN (1)
(1) National Taiwan University, Taipei, TAIWAN
There are evidences showing that Bacillus cereus effectively induces systemic resistance in Lilium formosanum and lily cv. Star Gazer. Differentially expressed genes of GRP1 (glycine-rich protein), MT1 (metallothionein-like protein), and PsbR (photosystem II) had been identified by suppression subtractive hybridization. Expression of these three genes in lily leaves increased in response to Botrytis elliptica-infection; but GRP1 and PsbR gene expressions decreased in the leaves of B. cereus-treated lily plants with or without subsequent B. elliptica-infection. In a cDNA-AFLP (amplified fragment length polymorphism) analysis of B. elliptica-infected leaves and the leaves of B. cereus-treated lily plants with or without subsequent Botrytis inoculation, over 100 cDNAs were cloned without the electrophoresis step. Sequence analysis indicated that most of the cloned cDNAs were metabolism and chromosome-related, and some were signal transduction, cell defense, transport, energy, protein synthesis, and transcription-related. Among them, four were analyzed by semi-quantitative RT-PCR to show the differential expression patterns under conditions of pathogen attack, rhizobacterium treatment and induced systemic resistance. B. cereus-treatment and B. elliptica-infection could increase gene expressions of GTPase-binding protein, calmodulin, and glutamine synthetase; however, expressions of these genes gradually decreased after Botrytis inoculation on B. cereus-treated lily plants. Since gene expression of glutamine synthetase could be induced by abscisic acid but suppressed by salicylic acid, and the fact that B. cereus-treatment suppressed gene expression of pathogenesis-related protein 1, it is presumed that the signaling pathway of B. cereus-induced systemic resistance is different from that of SAR in lily. Thus, our approach facilitates the identification of expressed sequences and the exploration of underlying mechanism of induced systemic resistance in the non-model system.
Ophiostoma and Geosmithia spp. associated with western oak bark beetle damage on declining California black oak and coast live oak in southern California
S. C. LYNCH (1), A. Eskalen (3), P. Zambino (4), T. Scott (2)
(1) Center for Conservation Biology, University of California, Riverside; (2) Department of Earth Science, University of California, Riverside; (3) Department of Plant Pathology and Microbiology, University of California, Riverside; (4) USDA Forest Service, Pacific Southwest Region, San Bernardino, CA
California black oak (Quercus kelloggii) and coast live oak (Quercus agrifolia) have undergone severe decline in San Diego County, California, since 2002. During a survey of San Diego and Riverside County oak stands in 2009–2010, western oak bark beetle (WOBB; Pseudopityophthorus pubipennis) galleries were abundant on branches of both species. Some bleeding and cankers in the cambium of boles were also associated with WOBB galleries. Fungi regularly recovered on cycloheximide-streptomycin MEA and tetracycline PDA from necrotic WOBB-infested cambium tissues were identified as Ophiostoma sp. and Geosmithia sp. by ITS4/5 rDNA sequencing. Pathogenicity of both fungi is being tested by inoculations in field trees to determine if these fungi could play a role in the decline and mortality of oak species in southern California within or outside the goldspotted oak borer (GSOB, Agrilus coxalis) zone of infestation.
Another step closer to implementing inoculum detection as a method to time initiation of fungicide applications for management of grape powdery mildew
W. F. MAHAFFEE (3), G. G. Grove (2), D. Martin (3), A. S. Albrecht (1)
(1) Dept. Plant Pathology, Oregon State University, Corvallis, OR; (2) Dept. Plant Pathology, Washington State University, Prosser, WA; (3) USDA-ARS Hort. Crops Res. Lab, Corvallis, OR
Inoculum detection for timing fungicide applications against grape powdery mildew has been shown to work using qualitative and quantitative PCR approaches. However, these approaches require expensive equipment, specialized skills, and labor costs that impede implementation by viticulturist. Loop mediated isothermic PCR (LAMP) is a robust method for the detection of DNA that can be performed with minimal equipment and skill. A set of LAMP primers were designed against the ITS2 segment of the ribosomal DNA region of Erysiphe necator that are specific and can detect less than one spore or less than 5 copies of target DNA in a purified plasmid. Spores were trapped from vineyard air using by continuously running an impaction trap with 40 × 1.5 mm stainless steel rods coated with vacuum grease and replacing sample rods every 3 to 4 days. DNA extraction was accomplished by placing rods in 100 µl of TE buffer, centrifuging for 1 min, boiling for 5 min, vortexing for 10 sec and then placing 5 µl DNA extract in PCR tube with mastermix. The PCR tube was then placed at 65°C for 45 min followed by 80°C for 5 min. Positive detection was determined by the formation of white precipitate. Grower implementation was tested by placing 3 traps at each vineyard with one processed by the grower using LAMP and the other processed in the LAB for LAMP and quantitative PCR. Grower implementation is being tested by placing 3 traps at each vineyard with one processed by the grower using LAMP and the other processed in the lab for LAMP and quantitative PCR. The results of the grower implementation will be presented.
Comparison of biofungicides and boscalid for management of Sclerotinia drop of lettuce
M. E. MATHERON (1), M. Porchas (1)
(1) The University of Arizona, Yuma, AZ
In Arizona, Sclerotinia drop of lettuce is caused by Sclerotinia minor and S. sclerotiorum. A field trial was conducted during the 2009 growing season to compare the level of disease control achieved with several different biofungicides and the conventional fungicide boscalid (Endura). Lettuce was seeded on raised beds in double rows 30 cm apart. At seeding, approximately 2,100 sclerotia of S. minor or 800 sclerotia of S. sclerotiorum produced in the laboratory were distributed on the surface of each 7.6-m-long plot between the rows of lettuce, then incorporated into the top 5-cm layer of soil. Products were applied to the bed surface at seeding, before initiation of sprinkler irrigation to germinate seed, and one or more times after lettuce was thinned. Compared to nontreated plots, the mean number of diseased plants at crop maturity in plots containing S. minor was reduced 72% by Endura, 70% by Contans, 50% by Humega, 33% by Silmatrix and SoilGard, 27% by Actinovate and 15% by Tenet. In plots infested with S. sclerotiorum, the number of diseased plants was reduced 96% by Contans, 76% by Endura, 48% by Humega, 46% by Actinovate, 32% by SoilGard, 28% by Silmatrix and 26% by Tenet.
Resident biology restricts proliferation of Macrophomina phaseolina in brassicaceae seed meal amended soil
M. MAZZOLA (1)
(1) USDA-ARS, Wenatchee, WA, U.S.A.
M. phaseolina is a pathogen of emerging importance in strawberry production systems. Studies were conducted to assess the efficacy of brassicaceae seed meal amendments for control of this pathogen and to determine the relative importance of soil biology and chemistry in any observed disease suppression. Seed meals were sourced from Brassica napus (canola), Sinapis alba (white mustard) and Brassica juncea (oriental mustard). When inoculated with M. phaseolina all seed meal amended soils limited persistence of the pathogen relative to the non-treated control. This was observed irrespective of whether a biologically active chemistry (e.g. allyl isothiocyanate by B. juncea) was produced in response to the seed meal amendment. When assays were conducted in pasteurized soils infested with the pathogen, all seed meal amendments failed to suppress M. phaseolina. Seed meals also effectively suppressed disease development when strawberry was planted in a soil naturally infested with M. phaseolina. However, disease suppression was temperature sensitive, and exhibited failure as soil temperature was elevated above 32°C. Exposure to AITC emitted from B. juncea amended soils for up to 8 h had a fungistatic but not fungicidal effect on M. phaseolina. These findings indicate that the resident soil biology is the dominant mechanism contributing to suppression of M. phaseolina in response to brassicaceae seed meal amendments.
Particle size affects Brassica juncea seed meal-induced pathogen suppression of Rhizoctonia solani AG-5
M. MAZZOLA (1)
(1) USDA-ARS, Wenatchee, WA, U.S.A.
R. solani AG-5 is a component of the pathogen complex that incites apple replant disease, and is suppressed via multiple mechanisms in response to B. juncea seed meal (SM) amendment. Allyl isothiocyanate (AITC) functions in suppression of this pathogen during the initial 24 h period post-seed meal amendment, but thereafter soil biology and specifically resident Streptomyces spp. play the dominant functional role in disease suppression. AITC emission was initiated earlier and reached higher maximal concentrations in soils amended with fine particle (<1 mm dia) than coarse particle (2-4 mm dia) SM. This corresponded with the level of disease suppression obtained when R. solani AG-5 and SM were introduced concurrently into soils and plant to apple; fine particle but not coarse particle SM suppressed apple root infection when applied to soil at a rate of 0.3% (wt/wt). Both fine and coarse particle SM amendment elevated resident Streptomyces approximately an order of magnitude by eight weeks post-application. When soil was infested with R. solani AG-5 subsequent to this eight week incubation period and planted to apple, both SM types effectively suppressed Rhizoctonia root rot. These findings demonstrate that at the rates utilized particle size will affect the efficacy of chemistry-based, but not biologically-based, suppression of R. solani in response to B. juncea SM soil amendment.
Verticillium dahliae genes putatively involved in microsclerotia development
N. P. MORALES (1), K. F. Dobinson (2)
(1) Department of Biology, University of Western Ontario, London, Ontario, CANADA; (2) Agriculture and Agri-Food Canada, London, Ontario, CANADA
Verticillium dahliae is a soil borne fungus, causal agent of an economically significant vascular wilt disease. V. dahliae produces persistent resting structures, known as microsclerotia, which are the primary source of disease inoculum in the field. Microsclerotium development has been studied at the morphological level, but little is known about the molecular mechanisms that govern this process. Recent gene expression studies and analysis of the V. dahliae genome sequence have revealed a diverse number of genes that may be involved in the microsclerotia development. This study focuses on the characterization of several class II hydrophobin genes, and a gene (provisionally designated VdHyp04) that encodes a hypothetical protein. Bioinformatics analyses revealed signal peptide and cleavage sites in all of the hydrophobin-like proteins as well as in VdHyp04, suggesting that all of these proteins are secreted. A methodology that depends on Agrobacterium tumefaciens-mediated transformation is being used to create knockout mutants for these genes. The system involves the transfer into a wild-type Verticillium strain of a non-functional copy of the gene of interest, and its subsequent homologous integration into the fungal genome to replace the wild-type locus. Transformants are being screened to identify strains in which the mutant gene has replaced the wild-type gene by homologous recombination. Morphological and molecular analyses of these mutants will be done to determine if these genes are involved or not in the microsclerotia development process, and data from these studies will be presented.
Genetic diversity within a vegetative compatibility group of aflatoxin-producing fungi
A. ORTEGA-BELTRAN (1), L. Grubisha (2), P. J. Cotty (2)
(1) The University of Arizona, School of Plant Sciences Tucson, AZ, U.S.A.; (2) USDA-ARS, School of Plant Sciences, Tucson, AZ, U.S.A.
Aflatoxin contamination of maize by Aspergillus flavus occurs frequently in both tropical and subtropical climates. Consumption of contaminated commodities adversely influences the health of both humans and domestic animals. Communities of Aspergillus flavus from maize field soils in the State of Sonora, Mexico were dominated by a single vegetative compatibility group (VCG SON003) in 2006. Communities’ structures of aflatoxin-producing fungi have been studied for decades but similar dominance has not previously been reported. Soil samples were collected in 4 agroecological zones across 300 km at elevations ranging from 6 m to 2100 m. In 2007 and 2008, the presence of VCG SON003 was drastically reduced. SON003 is also present in fungal communities resident in the U.S.A., where it has been reported from several states since 1987. Microsatellite markers were used to assess genetic diversity within SON003 before, during and after its dominance in 2006. 292 SON0003 isolates were screened for toxin-producing ability; 99% of isolates produced an average of 5789 µg/kg of aflatoxins. Results suggest explosion and dominance by a single clone of SON003 not detected prior to 2006. Implications of these observations on our understanding of the population biology of Aspergillus flavus will be discussed.
Biofungicides as transplant and soil treatment in the control of Phytophthora blight on chile pepper
S. SANOGO (1), L. Liess (1)
(1) New Mexico State University, Las Cruces, NM
Phytophthora capsici is a serious pathogen of a wide array of vegetable crops worldwide. On chile pepper (Capsicum annuum), P. capsici affects all plant parts, and commonly causes plant wilt as a result of severe damage inflicted to the root system. Management of Phytophthora blight requires a system approach. Control of this disease by use of biorationals is an emerging area of research. This study was conducted to determine the effects of transplant and soil treatment with biofungicides on the development of Phytophthora blight on chile pepper. Transplants (6-to-8 leaf stage) of AZ-20, a chile cultivar susceptible to P. capsici, were immersed in 0.1% suspension of two biofungicides (Actinovate and Mycostop Mix) and water (control) three days prior to transplanting into 12-cm round plastic pots filled with sterilized Terra-Lite Metro Mix 360. Soil was treated by drenching with the following products: 0.1% Mycostop Mix, 0.1% Actinovate, Ridomil Gold EC (1.16 liter/ha), and water (control). Three days following soil drenching, plants in all treatments were inoculated with an isolate of P. capsici at inoculum concentration of 10,000 zoospores per pot. Plants were monitored regularly, and disease severity was assessed weekly for six weeks. Disease severity was not significantly affected when transplants were treated with Actinovate and Mycostop Mix. Across all transplant treatments, soil treatment with Ridomil Gold EC significantly reduced disease severity over 50% compared to control. An effective soil treatment that reduces soil inoculum potential is essential in the management of P. capsici.
Characterization of VvBsl-1 and VvBsl-2 genes of Vitis vinifera in response to Botrytis cinerea infection
J. SERRANO-ACEVEDO (1), D. Herrera (1), P. Arce-Johnson (1)
(1) Department of Molecular Genetics and Microbiology, Biological Sciences Faculty, Pontificia Universidad Católica de Chile, Santiago, Chile
Chile is one of the most important grapes-exporting countries in the world. For this reason, grapes quality and pathogens control are always required. One of the most relevant grapevine pathogens is Botrytis cinerea, a necrotrophic fungus that produces the gray mould disease. The molecular mechanisms involved in gray mold disease development are poorly known. The objective of this work is to understand this interaction at the transcriptional level. Using a bioinformatic approach, we identified two gene models (named VvBsl-1 and VvBsl-2) with high identity to one Arabidopsis thaliana gene required for susceptibility to Botrytis cinerea. These genes are transcription factors of the R2R3MYB Family and they are grouped with Arabidopsis genes clade that response to Botrytis cinerea and ABA. It is known that members of this family are required to regulate important processes of the berry development, such as sugar and anthocyanin biosynthesis and antioxidants production. Both genes are induced during different stages of Cabernet Sauvignon berries development. We analyzed the expression of these genes in Cabernet Sauvignon leaves by Real time PCR. We found that VvBsl-1 and VvBsl-2 are highly induced by infection of Botrytis cinerea at 40 hours post-inoculation when compared to non inoculated leaves. Additionally, we analyzed the expression of these genes in others cultivars such as Kyhojo, Fue Kue and rootstocks, in Botrytis cinerea infected leaf and berries at different fruit development stages, as well as uninfected tissue.
Effects of fungicides on a mycophagous coccinellid may represent integration failure in disease management
A. M. SUTHERLAND (2), W. D. Gubler (2), M. P. Parrella (1)
(1) Department of Entomology, University of California, Davis, CA, U.S.A.; (2) Department of Plant Pathology, University of California, Davis, CA, U.S.A.
The adults and larvae of halyziine coccinellids (Coleoptera: Coccinellidae: Halyziini) are obligate mycophages on hyphae and conidia of powdery mildew (PM) (Erysiphales) fungi, plant parasites warranting chemical control in many managed systems. These insects have been observed to reduce PM severity through consumption. Fungicide applications, however, may interfere with this ecological service. Five commercial fungicides were topically applied to the mycophagous coccinellid Psyllobora vigintimaculata in the laboratory to gauge contact toxicity. In order to detect interference in the field, population density of naturally occurring P. vigintimaculata was assessed weekly in a northern California vineyard (Vitis vinifera, cultivar “Chardonnay”) over three years in relation to PM (Erysiphe necator) severity and in the presence of various fungicides. Wettable sulfur was toxic to adults in the laboratory, resulting in complete cohort mortality 24 hours after application. Topical applications of a strobilurin fungicide (trifloxystrobin) and a demethylation inhibitor fungicide (myclobutanil) also resulted in significant adult mortality. Rapid and complete larval mortality was observed in the laboratory after contact with wettable sulfur and myclobutanil. There was no effect on survival after contact with the PM-antagonistic bacterium Bacillus subtilis. Vineyard density of P. vigintimaculata was reduced in vines receiving applications of sulfur, myclobutanil, and several stobilurins, even when considering the covariate PM severity. The microbial antagonist Streptomyces lydicus did not significantly affect insect density. This study questions the integration of chemical disease management with naturally occurring mycophagous agents in some agricultural systems.
Influence of thrips control programs on TSWV incidence in Fresno County processing tomatoes
T. A. TURINI (2), M. Le Strange (1), R. L. Gilbertson (3)
(1) UC Coop. Ext., Tulare; (2) UC Coop. Ext., Fresno, CA; (3) UC Davis, CA
Tomato spotted wilt virus causes substantial losses in California processing tomatoes. Insecticide programs are used to limit disease spread by killing the thrips vector, primarily Frankliniella occidentalis, but the benefit of these programs has not been documented under San Joaquin Valley conditions. A field study was conducted in 2009 to evaluate affect of soil- and foliar-applied insecticide programs on incidence of TSWV. On 14 May, processing tomato plants cv. H 8004 were transplanted at a research center. A four replication split block design was used. Main plot treatments a) Platinum on 3 Jun, b) Platinum on 3 Jun followed by Venom on 7 Jul, and c) untreated control were applied through a buried drip irrigation system. Subplot treatments were the foliar applications: a) Radiant, Dimethoate EL, Lannate WP, and Radiant applied at 1 to 2 week intervals beginning 17 Jul, b) ‘treatment a’ without the first Radiant application, c) ‘treatment a’ without the last Radiant application and d) untreated control. Twenty-five flowers per plot were collected over time and numbers of thrips were recorded. Plants were carefully inspected for TSWV-symptoms and representative samples were confirmed with TSWV immunostrips. Results show that foliar applications reduced thrips counts, but the drip applications did not have a significant effect. Percent disease incidence on 2 Sep was similar among the three foliar treatments (23.5, 20.3 and 23.2) and significantly lower than the untreated control (33.2). Although only one season of data is presented, it suggests that foliar applications play a role in a TSWV management program.
Characterization of the powdery mildew fungus occurring on evergreen Rhododendrons in the Pacific Northwest
L. S. TYMON PUTNICKI (1), D. A. Glawe (1)
(1) Washington State University, Pullman, WA, U.S.A.
Powdery mildew is a disfiguring disease of deciduous and evergreen Rhododendron species. Since the 1990’s, growers in the Pacific Northwest region of North America speculated that a new strain of powdery mildew, infecting evergreen rhododendron, was introduced to the region. In order to test this hypothesis, morphological features of powdery mildew fungi infecting Rhododendron were characterized microscopically and phylogenetic analyses of ITS, 28S, and RPB1 sequences were performed. Specimens were collected from diseased ericaeous plants in botanical gardens and residential gardens in Oregon, Washington, and British Columbia. Anamorphs infecting evergreen rhododendron were morphologically similar to those infecting deciduous azalea, with kinked foot cells and singly-formed conidia. Collections from deciduous Rhododendron could be assigned to Erysiphe azaleae or E. vaccinii on the basis of morphological features, including chasmothecial appendages. Maximum Parsimony and Maximum Likelihood analyses of sequence data suggested that E. azaleae may represent a paraphyletic group. Results were consistent with the hypothesis that powdery mildew of deciduous Rhododendron is caused by a taxon distinct from that infecting evergreen Rhododendron. However, these results suggested that the ITS, 28S and RPB1 sequences used are not sufficient to resolve, with confidence, phylogenetic and taxonomic groups within the deciduous Rhododendron-infecting Erysiphales.
Characterization of ATG8 autophagy gene homologs in Verticillium dahliae and Verticillium albo-atrum
S. M. VAN TWEST (2), S. J. Grant (1), J. Cucullo (2), K. F. Dobinson (1)
(1) Agriculture & Agri-Food Canada, London, Ontario, CANADA; (2) University of Western Ontario, London, Ontario, CANADA
Both Verticillium dahliae and V. albo-atrum are soil-borne fungal pathogens that cause vascular wilt in many economically important plant species. While V. dahliae produces melanised resting structures called microsclerotia (MCS) that can persist in soil for up to 15 years and pose a significant challenge to disease control, the closely related V. albo-atrum survives by forming dark resting mycelia (DRM). To identify candidate genes responsible for resting structure development, cDNA libraries were constructed from cells grown in two environments; i) a simulated xylem fluid medium where the fungus exhibits dimorphic growth, and ii) conditions that favour near-synchronous microsclerotia development. Sequences highly similar to genes encoding the well-characterized yeast macroautophagy marker ATG8 were identified in both cDNA collections, indicating a role for autophagy in Verticillium development. In other filamentous fungi, autophagy has been shown to be required for nutrient recycling during starvation, and also to be involved in cellular differentiation and accompanying developmental processes such as germination, sporulation, and infection structure formation. We are characterizing the ATG8 genes of V. dahliae (Vd) and V. albo-atrum (Va). ATG8 gene knockout mutants have been generated in both species, and comparative morphological studies between wild-type and knockouts have shown that VdATG8 is involved in conidiation, dimorphic growth, and microsclerotia formation in V. dahliae. No morphological defects were observed in VaATG8 knockouts. Intriguingly, stressing the fungus by increasing the temperature, results in restoration of the wild-type phenotype in the vdatg8 strains. Recent data from other morphological and microscopic analyses of the mutants and complemented strains will be described.
Relationship among kernel drydown rates, environmental factors and resistance to gibberella ear rot, fusarium kernel rot and common smut of corn
K. XIANG (2), L. M. Reid (1), X. Zhu (1)
(1) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, CANADA; (2) Maize Research Institute, Sichuan Agricultural University, Ya’an 625014, Sichuan, P.R. CHINA
Gibberella ear rot (Fusarium graminearum), fusarium kernel rot (F. verticillioides) and common smut (Ustilago maydis) are three ear diseases of corn which frequently occur in Canada and many other countries. These diseases reduce yield, quality and the two fusarium pathogens may contaminate the grain with mycotoxins. Many studies have indicated that there is a short window of time in which a corn ear can be infected and that this window is greatly influenced by the environment. We hypothesized that genotypes with faster kernel dry down rates would be less infected as they would ‘escape’ colonization. In 2008 and 2009, six corn inbreds of varying resistance to each disease and eight F1 hybrids between some of these inbreds were used in a study to investigate the relationship between kernel drydown rate (KDR), susceptibility to the three ear diseases and environmental factors (corn heat units, total rainfall and global solar radiation) from silking to eight weeks after midsilk. Six inoculation treatments were used: 1) F. graminearum, kernel inoculation; 2) F. graminearum, silk channel inoculation; 3) F. verticillioides, kernel inoculation; 4) F. verticillioides, silk channel inoculation; 5) U. maydis; and 6) a sterile water control. Ear moisture was measured at five and eight weeks after pollination, using an electronic probe and used to calculate KDR. Significant (P < 0.01) genotype and treatment effects were found for KDR and disease severity. Statistically significant correlations were found with KDR for all three diseases and all inoculation techniques with the highest correlation for KDR and kernel inoculation with F. verticillioides. Correlations were also found between symptoms and some of the environmental factors investigated. It was concluded that KDR does play a role in the severity of disease symptoms for these three diseases.
Characterization and overexpression of L-amino acid oxidase from Trichoderma harzianum ETS-323
C. Yang (1), C. Cheng (3), S. Liu (2), K. PENG (3)
(1) Institute of Life Science, Tzu Chi University, Hualien 97004, TAIWAN; (2) Department of Molecular Biotechnology, Da-Yeh University, Changhua 51591, TAIWAN; (3) Institute of Biotechnology, National Dong-Hwa University, Hualien, 97401, TAIWAN
In our previous studies, a putative L-amino acid oxidase (LAAO) that was secreted by Trichoderma harzianum ETS 323 has been suggested to involve in its antagonism with Rhizoctonia solani. However, research which has empirically investigated the biochemical properties of L-amino acid oxidase from T. harzianum ETS 323 is scant. Therefore, the aim of this study attempts to explore the essential feature of L-amino acid oxidase of T. harzianum ETS 323 including protein sequence, catalytic activity and specificity for amino acid substrates. Currently, the T. harzianum ETS-323 LAAO (Th-LAAO) gene was overexpressed and purified from Escherichia coli. To our knowledge, this is the first overexpression of LAAO from Trichoderma. The amino acid sequence alignment analysis using the ClustalW revealed that Th-LAAO exhibited a highest identity (98%) with the T. harzianum mRNA for putative LAAO. The purified protein showed a molecular mass of 52 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and catalyzed H2O2 formation from the L-phenylalanine and L-histidine, thus confirming its LAAO activity, where the specific constant (kcat/Km) value of each was 0.11 and 0.06, respectively. To conclude, this study may be of importance in providing researchers with a better understanding of LAAO’s biological function from Trichoderma.
Species of Neofabraea responsible for anthracnose canker of apple trees in western Washington State
R. Zang (2), L. Huang (1), C. L. XIAO (2)
(1) Northwest A & F University, Yangling, Shaanxi, CHINA; (2) Washington State University, TFREC, Wenatchee, WA, U.S.A.
To determine species of Neofabraea responsible for apple anthracnose canker in western Washington, apple twigs with symptoms of anthracnose canker were collected in November 2007 from seven apple orchards in the Bellingham and Mt. Vernon areas, WA. A total of 146 isolates of putative Neofabraea spp. were obtained from the disease samples. All isolates were single-spore cultured. Total genomic DNA was extracted from cultures of these isolates. Amplification of DNA was performed in a multiplex PCR using four species-specific primers to identify isolates to species. Of the 146 isolates identified, 142 were N. malicorticis, 2 were N. alba, and 2 were Cryptosporiopsis kienholzii (the anamorph of a Neofabraea sp.). No N. perennans was found among the isolates tested. To test pathogenicity of these species on apple, 2-year-old twigs of Jonagold apple were wounded and inoculated with one representative isolate each of N. malicorticis, N. alba and C. kienholzii. Twig inoculations were conducted twice (late October and late November 2008) in the orchard. Sizes of canker were measured 2 or 3 months after inoculation. All three species were able to cause cankers on inoculated twigs, and the same fungi were re-isolated from diseased twigs. The results indicate that N. malicorticis is the major cause of anthracnose canker of apple trees in western WA and that N. alba and C. kienholzii also are able to cause cankers on apple trees.
Pathogenic races of Exserohilum turcicum on corn in Ontario and Québec
X. ZHU (1), L. M. Reid (1), T. Woldemariam (1)
(1) Eastern Cereal and Oilseed Research Centre, Agriculture and Agri-Food Canada, Ottawa, Ontario, CANADA
Northern corn leaf blight (Exserohilum turcicum = Helminthosporium Turcicum) incidence in Canada has increased from 2002 to 2009. Incidence in surveyed corn fields reached 65.5% in Ontario in 2009 and 72.7% in Québec in 2008. Evidence was also found indicating that ‘resistant’ genotypes were expressing susceptible lesions. To identify the pathogenic races of E. turcicum that exist in these two provinces, 157 samples of diseased leaf tissue from 21 counties in Ontario and 39 samples from 16 counties in Québec were collected in 2005 and 2006, respectively. A single lesion from each sample was used for race identification. The lesion was divided into two halves, one was allowed to sporulate and used to inoculate greenhouse grown corn plants in the winter of 2006–2007; the other half was used to inoculate field grown corn plants in 2007. Six corn inbred lines, A619, A619Ht1, A619Ht2, A619Ht3, A632HtN, H102, were inoculated; these inbreds have the 0, Ht1, Ht2, Ht3, HtN, and Htm major resistant genes, respectively. In the greenhouse all of the 196 samples expressed on host either a resistant (R) lesion with a yellow margin which sometimes developed a gray centre or an elliptical, gray coloured susceptible (S) lesion; in the field, 195 sampled lesions expressed these symptoms. In the greenhouse, the ratio of R:S was 0:196, 161:35, 115:81, 90:106, 181:15 and 126:70 for the 0, Ht1, Ht2, Ht3, HtN, Htm hosts respectively; similarily the ratios in the field were 0:195, 23:172, 108:87, 94:101, 108:87 and 183:12, respectively. Ht1 did not show resistance in the field as it did in the greenhouse. We identified 25 pathogenic races in the greenhouse, five with the higher frequency and 21 races were identified in the field, seven of which were higher frequency. Thus the expressions of each resistant gene were very complicated. Further identifications are required.