Presentation on theme: "Shiv Hiremath, Kirsten Lehtoma, and Jenise M. Bauman"— Presentation transcript:
1 Shiv Hiremath, Kirsten Lehtoma, and Jenise M. Bauman SURVEY FOR THE PRESENCE OF PHYTOPHTHORA CINNAMOMI ON RECLAIMED MINED LANDS IN OHIO CHOSEN FOR RESTORATION OF THE AMERICAN CHESTNUTShiv Hiremath, Kirsten Lehtoma, and Jenise M. Bauman
3 Chestnut in Ohio Coal Mine Reclamation: Fast growth rateCompliment native rangeProvides a venue for assessment
4 Early Reports: C. dentata mortality Phytophthora species are causal agents of “ink disease”Its presence in the Appalachian region, has been noticed on chestnut seedlings (Rhoades et al., 2003, 2009; James 2012).Backcrossed chestnuts are not breed for resistance to chestnut ink disease.Jeffers 2009
5 History of Chestnut Ink Disease Prior to chestnut blight, several Phytophthoras were introduced to the U.S. ( s, Jeffers et al. 2009).Castanea spp. reported dying of unknown causes as early as 1824 (Crandall et al. 1945)Not reported until 1932 (Milburn and Gravatt 1932)Found in all throughout N. America including 28 States of the U.S.Crandall et al. 1945
12 Infection MechanismZoospore finds host Encysts Direct Penetration
13 Range of Phytophthora in N. America A survey of soils associated with oak species for the occurrence of PhytophthoraSoils sampled from bases of healthy and declining oak treesP. cinnamomi was the most frequently isolated species (69.4%) of the PhytophthorasThe absence of P. cinnamomi above the 40ºN latitudeBalci et al. 2007
14 Range of P. cinnamomi in Ohio Survey of Phytophthora species in white oak decline in southern OhioThe most common species was P. cinnamomiP. cinnamomi was more commonly isolated with wet soilsThe population densities of P. cinnamomi were significantly with declining oaksBalci et al. 2010
15 Study Objective:Survey reclaimed mine sites in southern Ohio for presence of P. cinnamomiCompared sites that were reclaimed under 5 years to those reclaimed 25 years priorCompared two Phytophthora sampling techniques: direct plating and leaf baiting
16 Study Sites YEAR LOCATION SITES METHOD 2008 Greendale, Ohio, Ora Anderson Park8Direct platingIronton District, WNF62009The Wilds, International Road4The Wilds, Lake TrailThe Wilds, Site 2Nelsonville, Ohio52011The Wilds, Site 17Leaf baitingThe Wilds, Forest Site C072New Straitsville, OH10
17 Ironton District, WNFThe Wilds, International RoadNelsonville, OhioThe Wilds, Lake Trail
18 Methods: Direct Plating Soil was collected at a depth of 4-5 inchesTen grams of soil were diluted in 100 ml dH2O and plated onto PAR(PH)-V8 (Johnson and Curl 1972)Plates were incubated in the dark at 22 oC and examined under a dissecting microscope for the presence hyphaeSubcultures were made on the same selective medium to obtain a pure culture.The fungus was allowed to grow for 3 weeks and harvested
19 Methods: Leaf Baiting250 g of soil from each site was mixed with 1.25 L deionized waterAmerican chestnut leaves were floated on the surface of the waterAfter 3-5 days, necrotic areas were examined for sporangiaThese areas were plated onto PAR(PH)-V8 plates as described above to obtain a pure culture (Balci et al. 2007)
20 Methods: Molecular Identification Final identification of the fungus was made by sequencing the internal transcribed spacer region of the ribosomal DNAPCR reactions were analyzed on a 0.7% agarose gelBands produced from the primer pair were isolated from the gel using the Geneclean (MP Biochemicals)Sequences were analyzed in the GENBANK using BLASTPrimerSequenceYph1F5’CGACCATKGGTGTGGACTTT3’Yph2R5’ACGTTCTCMCAGGCGTATCT3’Ycin3F5’GTCCTATTCGCCTGTTGGAA3’Ycin4R5’GGTTTTCTCTACATAACCATCCTATAA3’Phytophthora genus-specific(470bp)P. cinnamomi –specific(243bp)
22 Results: Wayne National Forest YearLocationP. cinnamomiPhytophthora2008Ora Anderson Park0 %Ironton District2009Nelsonville, Ohio2011New Straitsville, OH10 %P. cinnamomi was not detected. We found another species, P. citricola. In addition to Phytophthora, we also detected another plant pathogen belonging to the genus Pythium at one of the sites (Ironton District).
23 Results: The WildsYearLocationP. cinnamomiPhytophthora2009The Wilds, Grassland0 %The Wilds, Lake TrailThe Wilds, Site 22011The Wilds, Site 1The Wilds, Forest50 %We also found P. citricola in the forested site. This indicates that at least some of the Phytophthora species are present in these reclaimed lands. However, their presence was not common or widespread.
24 SummaryNorthern tip of the distribution of P. cinnamomi and the pathogen may have not be able to survive the freezing conditions.Since these were reclaimed lands, fresh top soil layer would have been added and pathogen may not have had time to relocate to these soils.It is not known how conducive the harsh soil conditions (low nutrients, pH extremes, toxic metals) are for the establishment of the pathogen.If this pathogen cannot survive in the mined lands, they would make highly suitable locations for the American chestnut restoration (Barton, et al, 2010).
25 Future Studies:Continue to evaluate hybrid seedlings for resistance to P. cinnamomi.Identify backcrossed lines with high levels of resistanceDifficulty is that resistance appears to be incompletely dominant and regulated by more than one gene (Jeffers et al. 2010).Pre-screening for soils that do not harbor P. cinnamomi.Proper site selection of areas that have proper drainageContinue experimental plantings that identify methods that improve establishment and long-term survival of chestnut.
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