Multiple species of Colletotrichum can cause bitter rot disease of apple, but the identities and relative representation of the species causing the disease in Kentucky are unknown. A total of 475 Colletotrichum isolates were collected from diseased apples in 25 counties and characterized both morphologically and by using various molecular approaches. Four morphotypes corresponded to reported descriptions of bitter rot species. Morphotype 1, distinguished by the production of a pink color on potato dextrose agar (PDA), orange conidial masses, and fusiform spores, was consistent with C. acutatum. Morphotype 2, which produced gray or white mycelial colonies with orange conidial masses and fusiform spores, was also similar to C. acutatum. Morphotype 3 had abundant gray mycelium and rounded spores and was identical to C. gloeosporioides. Morphotype 4 produced ascospores and resembled Glomerella cingulata. Species-specific polymerase chain reaction (PCR) indicated that both Morphotype 1 and Morphotype 2 belonged to the C. acutatum species complex, whereas Morphotype 3 and Morphotype 4 corresponded to the C. gloeosporioides complex. Multigene sequence analyses revealed that sample isolates belonged to several newly erected species within these species complexes. Morphotype 1 was identified as C. fioriniae, which resides within the C. acutatum species complex. Morphotype 2 was identified as C. nymphaeae, which is also a species within the C. acutatum species complex. Some isolates of Morphotype 3 were identified as C. siamense and some as C. theobromicola; both species are grouped within the C. gloeosporioides species complex. Morphotype 4 was identified as C. fructicola, which is also placed within the C. gloeosporioides species complex. C. fioriniae was the most common species causing bitter rot in Kentucky, comprising more than 70% of the isolates. Molecular fingerprinting using random amplified polymorphic DNA (RAPD) suggested that isolates within C. fioriniae belonged to a relatively homogeneous population, while isolates within C. siamense, C. theobromicola and C. fructicola were more diverse. Infectivity tests on detached fruit showed that C. gloeosporioides species-complex isolates were more aggressive than isolates in the C. acutatum species complex. However, isolates within the C. acutatum species complex produced more spores on lesions compared to isolates within the C. gloeosporioides species complex. Aggressiveness varied among individual species within a species complex. C. siamense was the most aggressive species identified in this study. Within the C. acutatum species complex, C. fioriniae was more aggressive than C. nymphaeae, causing larger, deeper lesions. Apple cultivar did not have significant effect on lesion development. However, Colletotrichum species produced more spores on Red Stayman Winesap than on Golden Delicious. Fungicide sensitivity tests revealed that the C. acutatum species complex was more tolerant to thiophanate-methyl, myclobutanil, trifloxystrobin, and captan compared to the C. gloeosporioides species complex. The study also revealed that mycelial growth of C. siamense was more sensitive to tested fungicides compared to C. fructicola and C. theobromicola. These research findings emphasize the importance of accurate identification of Colletotrichum species within each species complex, since they exhibit differences in pathogenicity and fungicide sensitivity.

Rhizoctonia blight (sensu lato) is a common and serious disease of many turfgrass species. The most widespread causal agent R. solani consists of several genetically different anastomosis groups (AGs) and subgroups. Though anastomosis or hyphal fusion reactions have been used to group Rhizoctonia species, they are time consuming and sometimes difficult to interpret. Anastomosis reactions are incapable of identifying isolates belonging to different AG subgroups within an AG. This study evaluated molecular techniques in comparison with traditional anastomosis grouping (AG) to identify and group isolates of Rhizoctonia. More than 400 Rhizoctonia isolates were collected from diseased turfgrass leaves from eight geographic areas in Virginia and Maryland. A random sample of 86 isolates was selected and initially characterized by colony morphology, nuclei staining and anastomosis grouping. Molecular identification was performed by analysis of rDNA-ITS region and DNA fingerprinting techniques universally primed PCR (UP-PCR) and amplified fragment length polymorphism (AFLP). The cladistic analysis of ITS sequences and UP-PCR fragments supported seven clusters. Isolates of R. solani AG 1-IB (n=18), AG 2-2IIIB (n=30) and AG 5 (n=1) clustered separately. Waitea circinata var. zeae (n=11), and var. circinata (n=4) grouped separately. A cluster of six isolates (UWC) did not fall into any known Waitea group. Most of the binucleate Rhizoctonia-like fungi (BNR) (n=16) grouped separately. AFLP grouping also largely agreed with the above results. However, UWC isolates clustered into two groups. Molecular analyses corresponded well with traditional anastomosis grouping by clustering isolates within an AG or AG subgroup together. UP-PCR cross-hybridization could distinguish closely related Rhizoctonia isolates to their infraspecies level. Genetically related isolates belonging to the same AG subgroups cross-hybridized strongly, while isolates of different AGs did not cross-hybridize or did so weakly. Sequence-characterized amplified region (SCAR) markers were generated from UP-PCR products to identify isolates of major pathogenic groups AG 1-IB and AG 2-2IIIB. Specific primer pairs successfully distinguished isolates of AG 1-IB and AG 2-2IIIB from isolates of other AGs. Sensitivity of Rhizoctonia species and AGs was tested in vitro to commercial formulations of iprodione, triticonazole and pyraclostrobin. W. circinata isolates were moderately sensitive to iprodione while isolates of R. solani and BNR were extremely sensitive. Isolates of AG 2-2IIIB showed less sensitivity to triticonazole than other Rhizoctonia isolates. W. circinata var. zeae isolates were moderately sensitive to pyraclostrobin while most of the other isolates were extremely sensitive. / Ph. D.

Submitted by Marlene Santos (marlene.bc.ufg@gmail.com) on 2017-09-29T19:13:24Z No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-10-02T13:35:40Z (GMT) No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-10-02T13:35:40Z (GMT). No. of bitstreams: 2 Dissertação - William Andrés López Arboleda - 2015.pdf: 1579315 bytes, checksum: 378fd0e65f3b2919d56630143950ed75 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2015-03-27 / Sclerotinia sclerotiorum is a phytopathogenic fungus that infects more than 400 plant species, including common bean. Genetic variability studies in connection with phenotypic traits of agronomic interest are important to drive the control strategies against this pathogen. The aims of this study were: to evaluate the genetic variability, fungicide sensitivity, aggressiveness and to determine the proportion of MAT (Mating Type) alleles of 79 isolates of S. sclerotiorum distributed in four populations from common bean. Two populations represented a single sampling location in two different times (2000 and 2013 growing seasons). To evaluate the fungicide sensitivity a cell viability test based on the alamarBlue dye using mycelial growth was standardized. Dose-response curves for fluazinam, procymidone and benomyl were estimated using this test and were compared with dose-response curves estimated by the mycelial growth inhibition on PDA plate and the FRAC (Fungicide Resistance Action Committee) protocol. Despite the differences to assess the fungicide sensitivity between the three methods, the dose-response curves showed similar trends for the three fungicides. The fungicide sensitivity assessment at the four populations showed low sensitivity to benomyl in the Planaltina population. Furthermore, this population presented a principally clonal population structure, with a haplotype represented by 18 out of 20 isolates. Significant population differentiation in all pairwise comparisons of phi, except the comparison between EV_2013-NH, was detected. Five genetically homogeneous groups were inferred by the DAPC analysis. No group was conformed by isolates from the four populations. Only two haplotypes between the two populations from the same sampling location were shared. The hypothesis of random mating was rejected at the four populations; however this hypothesis was not rejected at the two major populations inferred by the DAPC analysis. The screening of mating type locus showed a dominance of Inv+ isolates and a high proportion of Inv+/Inv- isolates (presumable heterokaryons). / Sclerotinia sclerotiorum é um fungo fitopatogênico capaz de colonizar mais de 400 hospedeiras, sendo o agente causal do mofo branco no feijoeiro. Estudos de variabilidade genética associados a características fenotípicas de interesse agronômico, como a sensibilidade a fungicidas, oferecem informações importantes para direcionar estratégias de controle sobre este patógeno. Os objetivos deste trabalho foram avaliar a variabilidade genética, sensibilidade a fungicidas e agressividade de 79 isolados de S. sclerotiorum distribuídos em quatro populações procedentes de culturas de feijoeiro em pivô central. Duas destas populações representaram um único local de coleta em duas épocas diferentes (2000 e 2013). Para avaliar a sensibilidade a fungicidas foi padronizado um teste de viabilidade celular baseado no corante alamarBlue® sobre o crescimento micelial em microplaca de 96 poços. Curvas de dose-resposta para os fungicidas fluazinam, procimidona e benomyl, usando um isolado de S. sclerotiorum, foram estimadas com este método, e comparadas com curvas de dose-resposta obtidas com os métodos de inibição do crescimento em placa e o proposto pelo Fungicide Resistance Action Committee (FRAC). Apesar das diferentes abordagens as curvas dose-resposta mostraram tendências semelhantes para os três fungicidas. A avaliação da sensibilidade a fungicidas nas quatro populações indicou uma alta insensibilidade ao benomyl na população de Planaltina. Por outro lado, a estrutura populacional foi principalmente clonal com um haplótipo representado por 18 dos 20 isolados desta população. Diferenciação populacional significativa foi detectada em todas as comparações par a par do phi, com a exceção da comparação EV-2013-NH. A analise DAPC identificou cinco grupos geneticamente homogêneos. Nenhum dos grupos esteve constituído por isolados das quatro populações. Só dois haplótipos foram compartilhados pelas populações EV_2000 e EV_2013. A hipótese de acasalamento aleatório foi rejeitada nas quatro populações, no entanto não foi rejeitada nas duas maiores populações sugeridas pelo DAPC. O screening do Mating type locus (MAT) mostrou uma prevalência de isolados Inv+ e uma alta proporção de isolados Inv+/Inv-