Trichoderma isolates from tropical environments induce resistance against Phytophthora capsici in Korean hot pepper H. Bae (6), D. P. Roberts (5), H. Lim (4), S. Park (3), C. Ryu (2), R. L. Melnick (1), B. A. BAILEY (6)(1) Department of Plant Pathology, Pennsylvania State University, University Park, PA, USA; (2) Korea Research Institute of Bioscience and Biotechnology, Daejeon, Korea; (3) National Institute of Agricultural Science and Technology, RDA, Suweon, Korea; (4) USDA/ARS/PSI/FNPRU, Beltsville, MD, USA; (5) USDA/ARS/PSI/SASL, Beltsville, MD, USA; (6) USDA/ARS/PSI/SPCL, Beltsville, MD, USA
Isolates of several Trichoderma spp. were collected from environments where Theobroma cacao is grown as potential biocontrol agents for cacao diseases. The diversity of isolates collected led us to consider if these isolates have biocontrol activity in the Phytophthora capsici and Korean hot pepper (Capsicum annuum) pathosystem. Six Trichoderma isolates were tested for endophytic, mycoparasitic, antimicrobial, and induced resistance capabilities. Isolates DIS 70a, DIS 219b, and DIS 376f were mycoparasites of P. capsici in plate culture while isolates DIS 70a, DIS 259j, and DIS 320c produced antimicrobial compounds. Pepper seedlings were grown in soilless mix colonized by Trichoderma and analyzed for endophytic growth on roots and stems. All 6 isolates internally colonized pepper roots but not stems. Expression of plant defense related pepper ESTs were evaluated using RNA from leaves and roots of 32 day old Trichoderma colonized peppers. Microarray analysis of pepper gene expression 36 to 72 h after inoculation with isolates DIS 259j and DIS 376f identified a large group of highly induced EST putatively involved in plant defense. In bioassays, isolate DIS 376f provided the most consistent protection against P. capsici. Using the described screens, it may be possible to identify multiple pathways for protection of Korean hot pepper Trichoderma spp. leading to a logical approach of combining isolates to maximize synergistic effects and increase biocontrol efficacy.
Does snoA (suppressor-of-nimO) antagonize a DNA damage checkpoint pathway controlled by Rad9/53BP1 and gamma-H2AX?J. R. BREWER (1), S. W. James (1)(1) Gettysburg College, Gettysburg, PA
In the fungus Aspergillus nidulans the Dbf4-dependent kinase (DDK) is composed of regulatory and catalytic subunits encoded by nimO(^Dbf4) and cdc7, respectively. nimO(^Dbf4) associates with cdc7p, activating the kinase and escorting it to origins of replication where it triggers DNA synthesis. A nimO mutation, nimO18, confers temperature sensitive cell cycle arrest in late G1, and at permissive temperature exhibits profound sensitivity to agents that cause double strand breaks (DSBs), such as Diepoxyoctane (DEO). We discovered a novel suppressor of nimO18, called snoA (suppressor-of-nimO). Intriguingly, loss of snoA substantially alleviates the ts-lethal and DNA damage-sensitivities of nimO18, indicating that snoA may act normally to inhibit nimO function and thereby restrain DNA synthesis in response to DNA damage. In a search for other DNA damage responses that may be inhibited by snoA, we discovered that DEO-sensitive defects in gamma-H2AX (H2AX-S129A) and Rad9(^53BP1) (?Rad9) are also partially relieved by loss of snoA. gamma-H2AX and Rad9(^53BP1) are components of an ATM-dependent DNA damage response pathway that responds to DSBs. In this study, we are assessing epistasis relationships between gamma-H2AX, Rad9, snoA, and ATM with the aim to determine if snoA influences gamma-H2AX and Rad9(^53BP1) in an ATM-dependent or ATM-independent manner. (Supported by grants from Gettysburg College to SWJ and JRB).
Life cycle of Uromyces salsolae, a candidate fungal biological control agent for Salsola tragusC. A. CAVIN (1), D. K. Berner (1), W. L. Bruckart (1)(1) USDA, ARS, FDWSRU, Frederick, MD, USA
Salsola tragus (Russian thistle, Chenopodiaceae) is a major weed pest in the western United States. An isolate of the rust fungus Uromyces salsolae from the Yasensky Spit in Russia is currently under evaluation as a candidate for biological control of S. tragus in a Biosafety Level 3 (BL-3) containment greenhouse facility. The life cycle of U. salsolae has been completed in greenhouse studies, demonstrating that it is macrocyclic and autoecious on Russian thistle. Plant inoculations were made with spores from each stage in the fungus life cycle, demonstrating their viability and role in the life cycle. Data will be included in a risk assessment of U. salsolae for biological control of S. tragus.
Greenhouse germination and characterization of Synchytrium solstitiale resting sporesF. M. ESKANDARI (1), W. L. Bruckart (1)(1) USDA, ARS, FDWSRU, Frederick, MD, USA
During evaluation of Synchytrium solstitiale for biological control of yellow starthisle (YST, Centaurea solstitialis), protocol was developed for germination of resting spores. Resting spores mature in large numbers 7 to 10 days after galls develop, and they are distinct morphologically from sori in galls. Resting spores are dark, single-celled, and embedded within leaf and petiole tissues. The protocol involved dried leaves containing resting spores. Leaves were surface sterilized for 10 min in 10% bleach and rinsed (3 ×) for 10 min in sterile distilled water (SDW). Resting spores were scraped out of plant tissue, placed on 2% water agar (WA) and incubated in the dark at 10 (night) and 15 (day) centigrade. After 10–25 days, some resting spores germinated and formed a round, yellow-orange vesicle (that becomes a sorus) on the outside. Further development of the sorus occurred only after individual germinated resting spores were picked off of the WA and placed in SDW with streptomycin (100 ppm) in the well of a hanging-drop slide. Prepared slides were placed in a moist chamber and incubated under conditions mentioned above. Opaque orange sori changed to translucent pink sporangia in 2 to 24 hours, and movement of zoospores was seen after that development. Sporangia eventually ruptured and zoospores were released in a manner similar to that of release from sporangia developing from sori in galls. YST inoculated with germinated resting spores with sori became infected following the same protocol for inoculating YST with galled leaf material. These results prove the viability of resting spores and suggest they are a functional part of the S. solstitiale life cycle. Characterization of resting spore germination should facilitate taxonomic treatment of S. solstitiale.
How does loss of snoA (suppressor-of-nimO) protect nimO (never-in-mitosis) mutants from genotoxic stress?L. A. GOEDEKE (1), S. W. James (1)(1) Gettysburg College, Gettysburg, PA
In Aspergillus nidulans, nimO(^Dbf4) is the regulatory subunit of Dbf4 dependent kinase (DDK), which acts to trigger DNA synthesis at origins of replication. The nimO18 mutation confers temperature sensitive cell cycle arrest at G1/S, and at permissive temperature exhibits profound sensitivity to agents that cause double strand breaks (DSBs), such as Phleomycin (PHL). We identified a nimO18 suppressor, called snoA (suppressor-of-nimO), by mutations that rescued nimO18 ts-lethality. Intriguingly, loss of snoA function also substantially protects nimO18 from PHL, restoring near-wild type PHL-resistance. By examining the response of nimO18 to acute versus chronic PHL exposure, I was able to determine that the PHL sensitivity of nimO18 results from a defect in DNA repair, rather than by disabling a DNA damage checkpoint. I am examining how replication and repair processes may be perturbed by examining the recovery of mutants after induction of the S phase DNA replication checkpoint via hydroxyurea (HU) block-release experiments. Surprisingly, although nimO18 grows ~20% more slowly than WT, nimO18 mutants recover more quickly from HU-induced S phase arrest, and loss of snoA in turn slows this recovery. I am using a similar approach to determine how nimO18 and snoA mutants recover from DNA damage incurred during S phase. (Supported by grants from Gettysburg College to SWJ and LAG).
Functional characterization of the HYR1 gene involved in detoxification of reactive oxygen species in the rice blast pathogenK. HUANG (1), N. M. Donofrio (1)(1) University of Delaware, Newark, DE, USA
Rice blast fungus, Magnaporthe oryzae, causes a serious disease in cultivated rice worldwide. When compatible interactions happen between plants and pathogens, the plant releases reactive oxygen species (ROS) to attack the pathogen and protect itself. The pathogen has its own mechanism to cleave those ROS. HYR1 gene (MGG_04476) is one such candidate and encodes a glutathione peroxidase (GSHPx) domain in M. oryzae. Its homologue in the yeast, Saccharomyces cerevisiae, is Hyr1/YIR037W and was reported to be a glutathione-dependent phospholipid peroxidase (PhGpx) that specifically detoxifies phospholipid peroxide. To characterize it in M. oryzae, we have successfully knocked out HYR1 gene using homologous recombination method. The knockout mutants did not show any abnormal phenotypes in spore germination or pathogenicity assays, but we observe increased levels of growth inhibition when grown in complete media containing 0.5 mM, 1.0 mM and 2.0 mM hydrogen peroxide, respectively. Moreover, in yeast, Hyr1 has been identified to sense and transfer the oxidative signal to the transcription factor Yap1 upon accumulation of H(2)O(2). So in order to figure out the pathway of how this gene interacts with other reactive oxygen species cleavage-related genes in M. oryzae, we are extracting RNA from the hyr1 mutants and trying to compare their gene expression levels upon hydrogen peroxide contact. Thus far, we can conclude that the HYR1 gene is important for growing allowing the fungus to grow in low levels of hydrogen peroxide, but this gene does not seem to play a role in spore germination or pathogenicity.
Chlorosis and necrosis associated with introgression of a barley oxalate oxidase gene into flue-cured tobaccoC. S. JOHNSON (2), E. A. Grabau (1), J. G. Jelesko (1)(1) Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Tech; (2) Southern Piedmont Agric. Res. & Ext. Center and Dept. of Plant Pathology, Physiology, and Weed Science, Virginia Tech
Rhizoctonia solani causes damping-off and sore shin on tobacco (Nicotiana tabacum) seedlings, while basidiospores of its teleomorph, Thanatephorus cucumeris, cause a foliar disease referred to as “Target Spot”. Oxalic acid production may be an important factor the initiation of these important tobacco diseases. Field experiments were conducted in 2007 and 2008 to compare target spot severity among 7 transgenic entries of flue-cured tobacco cultivar ‘K 326’ containing a barley oxalate oxidase gene with disease levels on the non-transformed wild-type. Both experiments included the 7 transformants and the non-transformed wild-type, and were replicated 4 times. The 2007 experiment was arranged in a randomized complete block design, while the 2008 test was arranged in a split-plot design in which main plots were composed of 2 harvest treatments. Plots were either harvested sequentially (typical for flue-cured tobacco) in 2008 or harvested once at the end of the season. The 7 transformants and the non-transformed wild-type were randomized within each harvest method. Target spot severity was extremely low on all entries in both trials, but in both years an unusual and severe foliar chlorosis and necrosis was observed among the transgenic lines, but not in the non-transformed wild-type. Symptoms were first observed near the topping stage of plant development and worsened as leaf was harvested. Chlorosis on 29 Aug 2007 was greater (P < 0.05) for transgenic entries 15-5, 7-2, and 7-4 compared to 8-4, 5-1, and 5-2. A 1 through 5 rating scale for necrosis was also greater on 11 Sep 2007 for these same three entries versus the transgenic entries 8-4 and 5-1. Similar trends were observed in 2008, and were consistent across the 2 harvest methods evaluated. The causes and impacts of these symptoms are currently unknown and are being investigated.
Mitigating deoxynivalenol contamination in hulless barley and fuel ethanol co-productsP. KHATIBI (1), C. A. Griffey (1), D. G. Schmale (1)(1) Virginia Tech, Blacksburg, VA, USA
Hulless barley (HLSB) is a new and emerging crop in Virginia, and may be an important source of fuel ethanol in the future. Dried distiller’s grains with solubles (DDGS), a co-product of fuel ethanol fermentation, are rapidly becoming one of the main sources of feed for domestic animals. Fuel ethanol production may concentrate mycotoxins such as deoxynivalenol (DON) in DDGS, posing a significant threat to domestic animal health. Our work aims to genetically engineer Virginia HLSB lines with reduced DON potential and thus provide a safe supply of DDGS for animal feed. We determined the DON potential of 20 Virginia HLSB lines, and a number of these lines demonstrated low levels of DON across three years of testing. We generated callus from 17 HLSB lines, and five of the lines were selected for further tissue culturing analyses and genetic transformation. We cloned TRI101, a gene encoding a 3-O-acetyltransferase responsible for the conversion of DON to 3-acetyl-DON, from four different species of Fusarium. We expressed these genes in vitro, and assessed their ability to detoxify DON in a series of feeding studies. We are currently moving TRI101 into five selected HLSB lines, and we plan to monitor decreases in DON in both raw grain and DDGS following fuel ethanol production using our genetically-engineered lines.
Identification and characterization of a MAS3-homolog from the rice blast fungus, which is potentially involved in a novel function in appressorial developmentS. M. MATHIONI (2), C. Rizzo (3), J. A. Sweigard (1), A. M. Carroll (1), N. M. Donofrio (2)(1) Dupont Stine Haskell Research Center, Newark, DE, USA; (2) University of Delaware, Newark, DE, USA; (3) WuXi AppTech, Inc. Philadelphia, PA, USA
Magnaporthe oryzae, the causal agent of the most threatening disease of rice, uses a specialized structure called an appressorium to infect its plant hosts. The appressorium further develops a penetration peg to invade the plant cell. Thus far, more than thirty genes have been identified that play a role in appressorial formation and development through targeted deletion studies. In order to identify more genes involved in M. oryzae pathogenicity, we undertook a global gene expression experiment using microarrays. Of the genes with interesting expression profiles, we are currently studying one with similarity to a MAS3 (Magnaporthe appressoria specific) virulence factor gene; this gene showed increased expression 72 hours post-inoculation of barley, and during carbon and nitrogen starvation conditions. Targeted replacement of the MAS3-similar gene resulted in mutants with decreased appressorial formation. Database searches with the sequence of this gene revealed the presence of a CAS (capsule-associated) transmembrane domain from Cryptococcus neoformans, a human pathogen. This gene also presented protein homology to two other previously described M. oryzae genes, termed GAS1 and GAS2, which were involved in pathogenicity by decreasing the ability of the fungus to invade the plant, however, these mutants showed no appressorial defects. Based on the contrast between the homology of this newly-identified gene as well as its phenotype compared to the GAS1 and GAS2 genes, we proposed that this gene is involved in a novel function, which affects appressorial formation and potentially pathogenicity.
Activation of plant defense genes of Theobroma cacao using endophytic Bacillus spp.R. L. MELNICK (1), B. A. Bailey (2), M. D. Strem (2), P. A. Backman (1)(1) The Pennsylvania State University, University Park, PA, USA; (2) USDA-ARS Sustainable Perennial Crops Lab, Beltsville, MD, USA
In cacao, increasing loss to diseases supported by interest in sustainable agriculture has lead to research on biocontrol options for reducing cacao diseases. Endophytic Bacillus spp. were isolated from superior cacao trees near Quevedo, Ecuador and screened as potential biological control agents of cacao diseases. One elite Bacillus spp. reduced witches’ broom disease over 18-months of field evaluations, while a second isolate reduced disease during the dry seasons. It is hypothesized that one mode of action for this disease reduction is induced resistance. The two Bacillus spp. were tested for their ability to activate plant defense genes of the susceptible Pound7 genotype. Surface sterilized seeds were treated with log 8.0 CFU/ml bacteria, then planted into a sterile soil mix in double magenta boxes. Boxes were maintained in a growth chamber and opened at onset of leaf flush. At maturation of the initial leaf flush, samples were collected to determine impact of endophytes on gene expression. Additional plants were sprayed with Phytophthora capsici zoospores (log 3.0 spores/ml) and harvested 24-hours later to determine impact on priming. Analysis of gene expression, using quantitative-PCR determined that endophytic colonization of cacao plants by native Bacillus spp. altered expression of some cacao defense genes. Data will be presented on the effects of endophytic colonization on cacao defenses and priming for plant defenses.
Preliminary evidence for mixed populations of Ca. Liberibacter species in Huanglongbing infectionsE. N. POSTNIKOVA (1), A. L. Stone (1), C. M. Wilson (2), D. J. Sherman (1), A. Sechler (1), E. L. Schuenzel (1), N. W. Schaad (1), W. Schneider (1), V. D. Damsteegt (1)(1) USDA-ARS FDWSRU, Fort Detrick, MD; (2) University Wisconsin, Madison, WI
Huanglongbing (HLB) is the most serious insect-transmitted disease of citrus in the world. Originally found only in Africa and Asia, it was discovered in Brazil and Florida in 2004 and 2005, respectively. Three Candidatus Liberibacter species, Ca. L. asiaticus (Las), Ca. L. africanus (Laf), and Ca. L. americanus (Lam) have been identified as causal agents. DNA was extracted and the ITS region was cloned from 29 different HLB samples from four continents (11 countries). Up to 50 clones per sample were sequenced. In 84 clones from seven single HLB samples from China, Indonesia, Japan, Philippines, Taiwan, Thailand, and Vietnam only Las was found. A single sample from India, contained 3 Laf and 24 Las clones. All 87 clones of three samples from South Africa were identified as Laf. In 168 clones of 13 samples from Brazil, 124 were identified as Las, 30 were identified as Laf, and 14 were identified as Lam. Single clones from India and Taiwan appeared to be recombinants of Las and Laf. Seventy-one of 72 clones from three samples from Florida were Las, with the lone exception being a Laf clone. These results demonstrate that multiple Liberibacter species can coexist in a single plant.
Detection limit of Phytophthora ramorum-infected Rhododendron leaves using the Cepheid SmartCyclerK. E. SECHLER (1), M. M. Carras (1), N. Shishkoff (1), P. W. Tooley (1)(1) USDA, ARS, NAA, FDWSRU, Fort Detrick, MD
The Sudden Oak Death pathogen, Phytophthora ramorum, has killed thousands of trees in the coastal forests of California and has affected numerous other plant species in nurseries by causing a range of symptoms from small lesions to plant death. The devastating impact of this pathogen has prompted quarantines to prevent pathogen spread and increased sampling to identify infected areas. Since lesions vary in their size and number, the goal of this study was to determine the smallest amount of tissue needed to consistently detect P. ramorum from Rhododendron ‘Cunningham White’ plants. Using a previously described mitochondrial based real-time PCR assay and a Cepheid SmartCycler®, DNA samples extracted from symptomatic and asymptomatic tissues were tested. Consistently reproducible results were possible with as little as 19.2 mm2 of tissue taken from the margin of a lesion. Increasing the amount of infected tissue for the DNA extraction did not seem to improve detection. Varied results were obtained for DNA samples extracted from asymptomatic leaf tissues.
Sporulation capacity of Phytophthora ramorum on northern red oak and chestnut oakP. W. TOOLEY (1), M. Browning (1)(1) USDA-ARS Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD
Branches from six 2 to 3-year old northern red and chestnut oak seedlings were dip-inoculated with ca. 5,000 sporangia/ml of P. ramorum isolate Pr-6 and incubated at 100% relative humidity in dew chambers for 6 days. Three plants were then used to assess sporangia production, while the other three plants were used to assess chlamydospore production. Sporangia production was evaluated by incubating infected seedlings in a mist chamber and collecting sporangia produced on four misted leaves per plant suspended over 15µ-diameter nylon mesh screens. Chlamydospore content of leaf disks (6 mm diameter) removed from diseased leaves following a one month incubation in a greenhouse was also determined. Chestnut oak exhibited significantly greater disease incidence and severity compared with northern red oak (P < 0.01). However, sporulation levels were observed to be much larger in northern red oak. Total sporangia production per plant was not significantly different between the two species but when adjusted by lesion area, northern red oak produced 2294 sporangia/cm(^2) compared with only 259 sporangia/cm(^2) for chestnut oak (P < 0.05). Mean chlamydospore production per 6 mm-diameter leaf disk also was significantly greater for northern red oak compared with chestnut oak (28 versus 1 chlamydospore per disk). Knowledge of P. ramorum sporulation capacity in relation to disease incidence and severity on Eastern U.S. oak species will help determine the potential for epidemic development should the pathogen be introduced.
Susceptibility of sprouted oak acorns to Phytophthora ramorum zoosporesT. L. Widmer (1), S. C. DODGE (1)(1) USDA/ARS-FDWSRU, Fort Detrick, MD
Phytophthora ramorum is a recently emerged pathogen having established in Europe and several western U.S. states, including California and Oregon. It has a wide host range and is a threat to forest ecology and the nursery industry. In California, coast live oak (Quercus agrifolia) is a major host in natural settings. Although P. ramorum has not established in the eastern U.S., artificial stem and foliar inoculations have demonstrated that native eastern Quercus spp. are susceptible when inoculated with sporangia. The purpose of this study was to determine if the primary roots of different Quercus spp. native to the eastern U.S. could be infected by P. ramorum zoospores, which could be released from sporangia into natural water run-off. Sprouted acorns of Q. rubra, Q. palustrus, Q. coccinia, Q. alba, Q. michauxii and Q. prinus were exposed to motile zoospores (3000/ml) of P. ramorum for 1, 6, or 24 h, rinsed in water to remove any nonattached cysts, and transplanted to potting soil. After 4 weeks, the roots were weighed, surface sterilized, plated on PARPH+V8 selective medium and incubated for 5 to 7 days at 20°C. Developing P. ramorum was identified visually based upon colony morphology and characteristic chlamydospores and sporangia. Results showed that the primary roots of all oak species tested were susceptible to P. ramorum zoospores, and that infection could occur when exposed for only 1 h to the inoculum. Root weights were not negatively impacted by exposure to P. ramorum after 4 weeks, regardless of the oak species (P = 0.746).
Efficacy of acibenzolar-S-methyl and fungicides for Fusarium wilt of watermelonX. Zhou (2), K. L. EVERTS (1)(1) Univ. of Maryland, Univ. of Delaware; (2) University of Maryland
A preliminary experiment was conducted in the greenhouse in the fall of 2007 to evaluate the efficacy of soil-applied treatments for management of Fusarium wilt in watermelon caused by Fusarium oxysporum f. sp. niveum. Fifteen treatments [14 fungicides and a systemic acquired resistance (SAR) inducer] or water were applied to potting mixture in pots containing watermelon seedlings. Azoxystrobin (Quadris), acibenzolar-S-methyl (Actigard, the SAR inducer), thiophanate-methyl (Topsin M), prothioconazole (Proline), metconazole, and ipconazole (Vortex) reduced Fusarium wilt incidence and severity. These six chemicals applied once at transplant were also evaluated in naturally infested watermelon fields in Delaware and Maryland in 2008, which had moderate and high levels of F. oxysporum f. sp. niveum, respectively. In Delaware, acibenzolar-S-methyl, thiophanate-methyl, prothioconazole, and ipconazole significantly reduced wilt at 2½ weeks after transplanting compared to untreated plants. These treatments also had the highest numerical vine length and plot vigor scores although there were no significant differences among treatments (P > F = 0.0551 and 0.0516, respectively). In Maryland, all treatments except azoxystrobin reduced wilt incidence 4 and 5 weeks after transplanting but not at 6, 7, or 8 weeks. Marketable fruit yields did not differ among treatments in either state. Metconazole caused phytotoxicity in all experiments.
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