CHARACTERIZATION OF RHIZOCTONIA SOLANI AND RHIZOCTONIA-LIKE FUNGI INFECTING VEGETABLES IN NEW YORK AND THEIR PATHOGENICITY TO CORN A Thesis Presented to the Faculty of the Graduate School of Cornell University In Partial Fulfillment of the Requirements for the Degree of Master of Science by Mana Ohkura May 2008 ABSTRACT Vegetable growers in New York have recently observed that the corn rotation is no longer effective in suppressing diseases caused by Rhizoctonia solani and Rhizoctonia-like fungi. To investigate this problem, R. solani and Rhizoctonia-like fungi were isolated from naturally infected vegetables in New York. Sixty-eight isolates were genetically characterized and their pathogenicity to corn was determined under greenhouse conditions. Sequence analysis of the rDNA internal transcribed spacer region inferred 26 isolates to belong to R. solani anastomosis group (AG) 2-2 and 19 isolates to belong to AG 4. Remaining isolates belonged to AG 1, AG 2-1, AG 5, AG 11, Ceratobasidium AG (CAG) 2, CAG 6, and Waitea circinata var. zeae. This is a first report of AG 11 and W. circinata var. zeae recovered from naturally infected vegetables in New York. Pathogenicity trials on corn showed that the majority of isolates are pathogenic to corn and isolates belonging to AG 2-2 exhibited high virulence and isolates belonging to CAG 2 exhibited low virulence. These results suggest that certain strains of R. solani and Rhizoctonia-like fungi infecting vegetables in New York have acquired the ability to infect corn. In particular, isolates of AG 2-2 have been previously confirmed to produce the sexual stage under field conditions, suggesting that these isolates may have evolved to infect corn through sexual recombination. In addition, snap bean was inoculated with isolates exhibiting variable virulence on corn and a potential correlation between virulence on corn and snap bean was observed. BIOGRAPHICAL SKETCH Mana Ohkura was born on July 13, 1982 in Kasugai, Japan. As a child, she lived in the USA, India, and Japan. In 2001, she entered University of California, Davis and pursued a major in Biological Sciences with a Fungal Biology and Ecology minor. Her enjoyable experience working in Dr. Tom Gordon’s lab and going mushroom hunting with Dr. Mike Davis got her very interested in Mycology and Plant Pathology. In 2005, Mana came to Cornell University and joined Dr. George Abawi’s program in the Department of Plant Pathology. At Cornell, she worked on Rhizoctonia solani and Rhizoctonia-like fungi that infects vegetables in New York. iii to Dad iv ACKNOWLEDGMENTS I would like to thank my committee members, Dr. George Abawi, Dr. Kathie Hodge, and Dr. Chris Smart, for their time and input during my time at Cornell University. George has taught me about plant diseases and issues that growers deal with in the field. I appreciate his suggestions and patience when I was not sure about where to go with my project. Kathie has taught me a lot of interesting facts about fungi and I very much appreciate her support when I was confused about my career. I would also like to thank her for mentoring me during my teaching assistantships which made me realize the fun of teaching. Chris has guided me through all of the molecular work. I greatly appreciated her advice when I was having problems with troubleshooting and her suggestions have significantly helped me complete my thesis. In addition to my committee members, I would like to thank members of the Abawi lab, John Ludwig and Beth Gugino, for their support. The Smart lab, Ning Zhang, Maryann Herman, Tanya Taylor, and Holly Lange, provided valuable technical assistance in the molecular characterization of isolates. I am also grateful to Dr. John Barnard for providing expert assistance in statistics. v TABLE OF CONTENTS Biographical Sketch Dedication Acknowledgments List of Tables List of Figures Chapter 1. Introduction Systematics of Rhizoctonia Biology and Control of Rhizoctonia solani and Rhizoctonia-like fungi Rhizoctonia solani and Rhizoctonia-like Fungi Infecting Vegetables in New York Page iii iv v vi vii 1 1 6 9 Chapter 2. Characterization of Rhizoctonia solani and Rhizoctonia-like Fungi Infecting Vegetables in New York and their Pathogenicity to Corn Introduction Materials and Methods Isolate Collection Molecular Characterization Phylogenetic Analysis Pathogenicity Evaluation on Corn in the Greenhouse Pathogenicity Evaluation on Snap Bean in the Greenhouse Statistical Analysis Results Phylogenetic Analysis Pathogenicity Evaluation on Corn in the Greenhouse Pathogenicity Evaluation on Snap Bean in the Greenhouse Discussion References 21 21 24 24 27 30 35 39 39 42 42 45 51 51 57 vi Table 1.1 Table 1.2 Table 2.1 Table 2.2 Table 2.3 Table 2.4 LIST OF TABLES Genera with Rhizoctonia-like anamorphs, and their phylogenetic affiliation. Page 2 Anastomosis group, subgroup, and cultural type of Rhizoctonia solani and their common hosts. 7 Rhizoctonia solani and Rhizoctonia-like fungi of New York characterized in this study. 25 Polymerase chain reaction primer combinations, cloning status, sequencing primer(s), and GenBank accession numbers for individual isolates. 28 Reference sequences used to characterize Rhizoctonia solani and Rhizoctonia-like fungi isolated in New York. 31 Results from corn and snap bean pathogenicity trials and inferred identification of individual isolates. 48 vii Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 LIST OF FIGURES Inoculation of corn seedlings. Incubation of corn seedlings. Disease rating scale used in the corn pathogenicity trial. Disease rating scale used in the snap bean pathogenicity trial. Morpological diversity of Rhizoctonia solani and Rhizoctonia-like fungi collected in New York. Neighbor-joining tree of reference sequences and collected New York isolates. Bayesian inference tree of reference sequences and collected New York isolates. Disease severity on corn caused by isolates collected in New York, when grouped by inferred anastomosis group, subgroup, or species. Page 36 37 38 40 41 43 46 52 viii CHAPTER 1 Introduction Species of the form genus Rhizoctonia are diverse and ubiquitous in the soil, often associated with plant roots. Some are mycorrhizal, most are saprobic, and many are economically important plant pathogens that occur globally and cause disease on a wide range of hosts (Garcia et al. 2006; Sneh et al. 1996). However, studying these fungi has been a challenge due to their ambiguous taxonomy (Cubeta and Vilgalys 1997). Systematics of Rhizoctonia The form genus Rhizoctonia traditionally includes filamentous soil fungi that do not produce asexual spores, possess brown pigmented hyphae, and possess rightangled branching points with constrictions. The use of such general vegetative features as taxonomic characters has resulted in a taxon that includes a heterogeneous mix of polyphyletic fungi (Garcia et al. 2006; Stalpers and Andersen 1996). These fungi produce a diversity of teleomorphs, but the difficulty in inducing the teleomorphs limited Rhizoctonia taxonomy to be primarily dependent on anamorph features (Garcia et al. 2006). Scientists have studied morphological and ultrastructural features, hyphal anastomosis reactions, and nuclear condition to better understand Rhizoctonia systematics (Andersen 1996; Moore 1987, 1996; Parmeter et al. 1967; Tu and Kimbrough 1978; Tu et al. 1977). The discovery of various teleomorphs has cleared up some of the taxonomic ambiguity and currently, members of the form genus Rhizoctonia sensu lato have been segregated into at least seven teleomorphic genera (Garcia et al. 2006; Tu and Kimbrough 1978). The teleomorphs within the form genus Rhizoctonia and their corresponding anamorphs are listed in Table 1.1. Most fungi in the form genus Rhizoctonia belong to the basidiomycetes; 1 Table 1.1 Genera with Rhizoctonia-like anamorphs, and their phylogenetic affiliation. Phylum Ascomycota Basidiomycota Teleomorph Tricharina Eckblad Ceratobasidium Rogers Waitea Warcup & P. H. B. Talbot Tulasnella Schroeter Sebacina Tulasne Thanatephorus Donk Helicobasidium Pat Anamorph Ascorhizoctonia Yang & Korf Ceratorhiza Moore Chrysorhiza T. F. Andersen & Stalpers Epulorhiza Moore Opadorhiza Moore Rhizoctonia J. G. Kuhn Thanatophytum Nees 2 however, some are ascomycetes (Moore 1987; Tu and Kimbrough 1978; Yang and Korf 1985). The genus Thanatephorus (Donk 1956) was erected for the teleomorph of R. solani ( = T. cucumeris), the most widely studied species in the form genus Rhizoctonia (Garcia et al. 2006; Stalpers and Andersen 1996). The anamorphic genus Moniliopsis Ruhland was previously proposed for Rhizoctonia-like species with Thanatephorus and Waitea teleomorphs (Moore 1987). However, since the name Rhizoctonia solani is well-established in the literature of plant pathology, a formal proposal to conserve R. solani as the type species for Rhizoctonia was made (Stalpers et al. 1998) and was approved at the 2005 International Botanical Congress (Vienna Code) (McNeill et al. 2006). Therefore, currently Rhizoctonia sensu stricto refers to anamorphs with Thanatephorus teleomorphs, including R. solani. The term “binucleate Rhizoctonia” is often used to describe Ceratobasidium species, which have Rhizoctonia-like anamorphs containing two nuclei in each cell. In following sections of this thesis, fungi within the form genus Rhizoctonia s. l. will be referred to as “R. solani and Rhizoctonia-like fungi” as the thesis will deal with specific groupings within R. solani and fungi outside of Rhizoctonia s. s. The most studied Rhizoctonia species, R. solani, is considered a species complex, comprised of many genetically distinct lineages. Hyphal fusion reactions have been used to recognize anastomosis groups (AGs) within the species complex (Anderson 1982). Isolates belong to the same AG if their hyphae grow toward each other and fuse; fused and adjacent cells may or may not remain alive. On the other hand, isolates belong to different AGs if their hyphae do not undergo fusion (Carling 1996; Vilgalys and Cubeta 1994). Currently, there are 14 AGs: AG 1 through AG 13 and AG-BI (Carling et al. 2002c). AG-BI consists of bridging isolates that each anastomose with more than one AG (Carling 1996). Presently, AGs 1, 2, 3, 4, 6, 8, 9, 11, 12 are further divided into subgroups based on additional criteria such as colony 3 morphology, genetic variability, biochemical properties, and pathogenicity (Carling 1996). AG subgroups are determined by different criteria depending on the particular AG. For example, subgroups within AG 1 (AG 1-IA to AG 1-ID) are based on colony morphology and pathogenicity, AG 2 subgroups (AG 2-1 to AG 2-4) are based on hyphal fusion frequency, AG 4 subgroups (AG 4-HGI to AG 4-HGIII) are based on DNA-DNA complementarity and fatty acid analysis, and AG 8 subgroups (AG 8-ZG1 to AG 8-ZG5) are based on zymogram patterns (Carling 1996; Carling et al. 2002b; Johnk and Jones 2001; Naito and Kanematsu 1994; Priyatmojo et al. 2001). In addition, AG 2-2 is further subdivided into cultural types (AG 2-2IIIB, AG 2-2IV, AG 2-2 LP) according to pathogenicity and cultural morphology (Hyakumachi et al. 1998; Ogoshi 1987). AG 2-2IIIB was identified on mat rush (Lomandra longifolia) and referred to as the rush type, AG 2-2IV was identified on sugar beet and referred to as the root rot type, and AG 2-2LP was identified on warm season turf grasses and referred to as the large patch type (Hyakumachi et al. 1998; Ogoshi 1987). Similar to R. solani, Ceratobasidium and Waitea species have been subdivided into anastomosis groups (CAG/AG and WAG respectively) as well (Ogoshi et al. 1983b; Oniki et al. 1985). In the United States, Ceratobasidium species are divided into CAG 1 to CAG 7, while in Japan, they are divided into AG A to AG U (Burpee et al. 1980a, 1980b; Hyakumachi et al. 2005; Ogoshi et al. 1983a; Ogoshi et al. 1983b). Characterizing R. solani and Rhizoctonia-like fungi by anastomosis reactions is time-consuming and can be ambiguous due to the presence of bridging isolates and isolates that have lost the ability to anastomose (Hyakumachi and Ui 1987; Sharon et al. 2006). Nutritional conditions can also affect anastomosis reactions (Yokoyama and Ogoshi 1988). Fortunately, the advance in molecular techniques has facilitated a more accurate classification of these fungi (Sharon et al. 2006). Various molecular markers have been developed to study different taxonomic levels of R. solani and Rhizoctonia- 4 like fungi (Cubeta and Vilgalys 1997; Johanson et al. 1998). In order to identify R. solani and Rhizoctonia-like fungi to AGs, currently DNA sequence analysis of the ribosomal RNA genes and in particular the internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA (rDNA) are considered to be appropriate (Gonzalez et al. 2001; Sharon et al. 2006). Studies have shown that it is possible to infer the species, AG, or subgroup of an unknown R. solani or Rhizoctonia-like fungus by constructing phylogenetic trees with rDNA ITS sequences of previously characterized isolates (Kuramae et al. 2003; Kuramae et al. 2007; Lehtonen et al. 2008; Manici and Bonora 2007; Rinehart et al. 2007). These studies have been conducted using isolates recovered from various crops as well as diverse locations suggesting the wide applicability of this protocol. The rDNA ITS sequence has also been used to study the phylogenetic relationships of already established AG, subgroups, and cultural types (Carling et al. 2002b; Gonzalez et al. 2006; Gonzalez et al. 2001; Kuninaga et al. 1997; Pope and Carter 2001; Salazar et al. 2000; Salazar et al. 1999; Sharon et al. 2006; Toda et al. 2004; Vilgalys and Cubeta 1994). Such studies have revealed contradictions between molecular and traditional systematics of R. solani and Rhizoctonia-like fungi: Two of these studies showed that subgroups within AG 2 are phylogenetically distant from each other, suggesting that anastomosis reactions are not good indicators of evolutionarily distinct units (Kuninaga et al. 1997; Vilgalys and Cubeta 1994). Another study showed that Ceratobasidium AGs may be polyphyletic in origin and that certain CAGs are more closely related to R. solani AGs (Gonzalez et al. 2001). The rDNA ITS sequence has also been used to characterize and propose new AGs and subgroups (Carling et al. 2002b; Carling et al. 2002c; Kuninaga et al. 2000). Thus, the rDNA ITS region appears to be useful in demonstrating phylogenetic relationships of R. solani and Rhizoctonia-like fungi. 5 Biology and Control of Rhizoctonia solani and Rhizoctonia-like fungi Rhizoctonia solani and Rhizoctonia-like fungi are geographically distributed worldwide and include some of the world’s most devastating plant pathogens. Different AGs and species have different host ranges and most vascular plants are potential hosts of plant pathogenic R. solani or Rhizoctonia-like fungi (Ogoshi 1996; Roberts 1999). Common symptoms caused by these fungi include damping off, root rot, stem rot, foliar blight, and stem canker (Agrios 2005). The widely studied species R. solani has a very wide host range and can infect vegetables, grasses, ornamentals, fruit trees as well as pine trees (Garcia et al. 2006; Gonzalez et al. 2006). Some AGs have a wide host range, while other AGs have a narrow host range (Ogoshi 1996). Table 1.2 summarizes the different AGs, subgroups, and cultural types and their corresponding common hosts (Garcia et al. 2006; Sneh et al. 1991). Many R. solani and Rhizoctonia-like fungi produce sclerotia that come in various shapes and sizes (Roberts 1999; Sumner 1996). Sclerotia are resistant, asexual propagules that allow survival in the soil for several years and can also serve as a source of inoculum (Roberts 1999; Sherwood 1970; Sumner 1996). In Thanatephorus species, “loose type” sclerotia are formed that lack a distinct rind (Tu and Kimbrough 1975). Under certain field conditions such as high relative humidity, T. cucumeris produces sexual propagules, known as basidiospores, that cause aerial infections. In contrast to asexual propagation by mycelia and sclerotia, disease spread is considered to be faster by basidiospores (Naito 1996). Furthermore, sexual reproduction can increase gene diversity through recombination and therefore is more likely to yield new combinations of virulence genes to overcome plant resistance (Agrios 2005). On the other hand, some R. solani and Rhizoctonia-like fungi are hypovirulent or nonpathogenic. These strains are studied for their potential use in biocontrol. Most of 6 Table 1.2 Anastomosis group (AG), subgroup, and cultural type of Rhizoctonia solani and their common hosts (Garcia et al. 2006; Mazzola et al. 1996; Sneh et al. 1991; Tu et al. 1996). 7 AG / Subgroup AG 1 IA AG 1 IB AG 1 IC AG 2-1 AG 2-2IIIB AG 2-2IV AG 2-3 Hosts rice, corn sorghum, bean, soybean, turfgrass, camphor seedlings, crimson clover bean, rice, soybean, leguminous woody plants, lettuce, hortensia, cabbage, figs buckwheat, carrot, soybean, flax, pine, lettuce Crucifers, strawberry, tulip, Japanese radish, subterranean clover rice, mat rush, ginger, turfgrass, corn, sugar beet, Chrysanthemum, Gladiolus, edible burdock, tree seedlings, soybean sugar beet, turfgrass Soybean AG 3 (PT, TB) potato, tabacco, tomato, egg plant, pepper AG 4 (HGI, HGII, HGIII) tomato, pea, potato, soybean, onion, cotton,snap bean, Loblolly pine seedlings, peanut, slash pine, cucumber, corn AG 5 potato, turfgrass, bean, soybeans, AG 6 (HG-I, GV) non pathogenic AG 7 AG 8 (ZG 1-1, ZG 1-2, ZG 1-3,ZG 1-4, ZG 1-5) AG 9 (TP, TX) Soybeans Poaceae, cereals crucifers, potato AG 10 AG 11 AG 12 AG 13 AG BI non pathogenic Wheat cauliflower, radish non pathogenic non pathogenic 8 these are Ceratobasidium species, but certain R. solani AGs are avirulent as well, such as AG 10 (Sneh 1998). Control of plant diseases caused by R. solani and Rhizoctonia-like fungi is difficult due to various biological properties of the pathogen. Their wide host ranges and versatility has made breeding for resistant cultivars difficult and their capability to adapt allows great potential for the pathogen to overcome ecological changes (Baker 1970; Leach and Garber 1970; Ogoshi 1996). The most popular control method is chemical control and growers highly depend on fungicides to suppress diseases caused by R. solani and Rhizoctonia-like fungi (Kataria and Gisi 1996). However, disease control using fungicides is not always effective. Satisfactory disease control can be hard to achieve due to the pathogens’ soil-borne nature, difficulty in timing of fungicide application, and taxonomic complexity among R. solani and Rhizoctonialike fungi (Katan 1996; Kataria and Gisi 1996; Olaya et al. 1994). In addition, even if a fungicide is known to be effective, growers will prefer not to use it if it is expensive (Abawi, personal communication). Therefore, crop rotation is commonly recommended as a method to control diseases caused by R. solani and Rhizoctonialike fungi (Huber and Sumner 1996). For vegetables, grain crop rotations are suggested to suppress diseases caused by R. solani (Leach and Garber 1970; Reiners and Petzoldt 2006). Rhizoctonia solani and Rhizoctonia-like fungi Infecting Vegetables in New York Rhizoctonia solani and Rhizoctonia-like fungi cause root and foliar diseases on various vegetables such as beans, table beets, carrots, and cabbage in New York State. The importance of the pathogen in New York has increased since the discovery of its perfect state, T. cucumeris on table beets in 1990 (Olaya and Abawi 1991). Subsequently, T. cucumeris was identified on snap beans and other crops in the state 9 as well (Abawi et al. 1995). The damage caused by R. solani and related fungi in New York has increased steadily during the past 10 years and the production of large numbers of aerially dispersed basidiospores is thought to be one of the contributing factors (Olaya and Abawi 1994b). Another factor is changes in cultural practices where large tractors throw infested soil onto crowns of the plants as they cultivate the field for weed control (Olaya and Abawi 1994b). Until recently, grain crop rotations were effective in suppressing vegetable diseases caused by R. solani and Rhizoctonia-like fungi (Abawi and Ludwig 2005; Reiners and Petzoldt 2006). However, during the past few years New York growers have reported that the corn rotation has been ineffective (Abawi, personal communication). A similar problem has been reported in Germany, where disease caused by R. solani on sugar beets has increased in locations where narrow rotations of sugar beet and corn are practiced. They demonstrated that the main strain known to be problematic on sugar beet in Europe, AG-2-2IIIB, has the ability to infect sugar beet after surviving on corn residues in the field (Ithurrart et al. 2004). Additionally, Win and Sumner (1988) have shown that disease on beans is more severe when beans are planted after corn that was infested with AG-4 and AG-2-2 (Win and Sumner 1988). Among R. solani and Rhizoctonia-like fungi, isolates belonging to AG 1, AG 2, AG 4, and W. circinata var. zeae are known to cause disease on corn (Garcia et al. 2006; Mazzola et al. 1996; Sneh et al. 1991; Sumner and Bell 1982a, 1982b). Within AG 1 and AG 2, subgroups AG 1-IA and AG 2-2 have been well documented to infect corn (Garcia et al. 2006; Li et al. 1998; Sneh et al. 1991; Sumner and Bell 1982b). Furthermore, cultural type AG 2-2IIIB has been reported to be the causal agent of root rot on corn in the United States (Ithurrart et al. 2004). Previous studies in New York have shown that the dominant AGs present in western New York are AG 2-2 on table 10 beet (88%) (Olaya and Abawi 1994a), AG-4 on snap bean (55%) (Galindo et al. 1982), and AG 1 on cabbage (Abawi and Martin 1985). Other isolates found associated with table beets belonged to AG-5, AG-4, AG-2-1, and binucleate Rhizoctonia (Olaya and Abawi 1994a), and those associated with snap beans belonged to AG-1 and AG-2 (Galindo et al. 1982). One possible explanation why the corn rotation is ineffective may be due to the existence of undetected R. solani subgroups or cultural types that are pathogenic on corn. Previous characterization in this region showed that AG and subgroups pathogenic on corn, AG 1, AG 2-2, and AG 4 are present. However, these studies were conducted more than 10 years ago and isolates were not identified to further subgroup or cultural type. If AG 1-IA and AG 2-2IIIB can be proven to be present, it would confirm the presence of well-documented strains pathogenic to corn. As for AG 4, specific subgroups known to be pathogenic to corn are not well established. A second possible reason for the ineffectiveness of the corn rotation may be that isolates that cause disease on vegetables have adapted to survive on corn. There have been two reports of AG-5, a group not commonly found infecting corn, causing infection on corn (Li et al. 1998; Tomaso-Peterson and Trevathan 2007). Conversely, isolates that usually infect corn may have acquired the ability to infect vegetables. For example, isolates of the Rhizoctonia-like fungus, W. circinata var. zeae are known to be pathogenic to corn (Mazzola et al. 1996; Sneh et al. 1991). However, W. circinata var. zeae has been found to cause disease on onions (Erper et al. 2006) and has been isolated from soybean and bean in Turkey (Erper et al. 2005). Lastly, a nonpathogenic AG or species may have emerged with the ability to infect these crops. For instance, AG-13, usually reported to be non-pathogenic, has been isolated from corn in Mississippi (Garcia et al. 2006; Tomaso-Peterson and Trevathan 2004b). Accurate assessment of present subdivisions of R. solani and Rhizoctonia-like fungi in 11 New York and their effect on corn may clarify some of the questions raised above. The aim of this study was to characterize the R. solani and Rhizoctonia-like fungi present in New York vegetable field soils using phylogenetic analysis of the rDNA ITS sequences, and to evaluate corn as a potential host. Results from these experiments can be used to test the following hypotheses to explain the ineffectiveness of the corn rotation: H1. Isolates of R. solani known to be pathogenic to corn, AG 1-IA and AG 22IIIB, exist in this region. H2. Isolates of R. solani and Rhizoctonia-like fungi that infect vegetables have gained the ability to infect corn. H3. Non-pathogenic isolates of R. solani and Rhizoctonia-like fungi have developed the ability to infect vegetables and corn. 12 REFERENCES Abawi, G. S., and S. B. Martin. 1985. Rhizoctonia foliar blight of cabbage in New York State. Plant Disease 69 (2):158-161. Abawi, G. S., and J. W. Ludwig. 2005. Effect of three crop rotations with and without deep plowing on root rot severity and yield of beans. Annual Report of the Bean Improvement Cooperative 48:118-119. Abawi, G. S., G. Olaya, and J. W. Ludwig. 1995. Occurrence of Thanatephorus cucumeris on snap beans in New York. Phytopathology 85 (12):1554. Agrios, G. N. 2005. Plant pathology Fifth edition ed. New York: Elsevier Academic Press. Andersen, T. F. 1996. A comparative taxonomic study of Rhizoctonia sensu lato employing morphological, ultrastructural and molecular methods. Mycological Research 100:1117-1128. Anderson, N. A. 1982. The genetics and pathology of Rhizoctonia solani. Annual Review of Phytopathology 20:329-347. Baker, K. F. 1970. Types of Rhizoctonia solani diseases and their occurrence. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press. Burpee, L. L., P. L. Sanders, H. Jr Cole, and R. T. Sherwood. 1980a. Anastomosis groups among isolates of Ceratobasidium cornigerum and related fungi. Mycologia 72 (4):689-701. Burpee, L. L., P. L. Sanders, H. Jr Cole, and R. T. Sherwood. 1980b. Pathogenicity of Ceratobasidium cornigerum and related fungi representing 5 anastomosis groups. Phytopathology 70 (9):843-846. Carling, D. E. 1996. Grouping of Rhizoctonia solani by hyphal anastomosis reaction. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. Jabaji-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Carling, D. E., S. Kuninaga, and K. A. Brainard. 2002a. Hyphal Anastomosis Reactions, rDNA-Internal Transcribed Spacer Sequences, and Virulence Levels Among Subsets of Rhizoctonia solani Anastomosis Group-2 (AG-2) and AG-BI. 13 Carling, D. E., R. E. Baird, R. D. Gitaitis, K. A. Brainard, and S. Kuninaga. 2002b. Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani. Phytopathology 92 (8):893-899. Cubeta, M. A., and R. Vilgalys. 1997. Population biology of the Rhizoctonia solani complex. Phytopathology 87 (4):480-484. Donk, M. A. 1956. Notes on resupinate Hymenomycetes - II. The tulasnelloid fungi. Reinwardtia 3 ((3)):363-379. Erper, I., G. Karaca, and I. Ozkoc. 2005. First report of root rot of bean and soybean caused by Rhizoctonia zeae in Turkey. Plant Disease 89 (2):203. Erper, I., G. H. Karaca, M. Turkkan, and I. Ozkoc. 2006. Characterization and pathogenicity of Rhizoctonia spp. from onion in Amasya, Turkey. Journal of Phytopathology 154 (2):75-79. Galindo, J. J., G. S. Abawi, and H. D. Thurston. 1982. Variability among isolates of Rhizoctonia solani associated with snap bean hypocotyls and soils in New York. Plant Disease 66 (5):390-394. Garcia, V. G., M. A. P. Onco, and V. R. Susan. 2006. Review. Biology and systematics of the form genus Rhizoctonia. Spanish Journal of Agricultural Research 4 (1):55-79. Gonzalez, D., M. A. Cubeta, and R. Vilgalys. 2006. Phylogenetic utility of indels within ribosomal DNA and beta-tubulin sequences from fungi in the Rhizoctonia solani species complex. Molecular Phylogenetics and Evolution 40 (2):459-470. Gonzalez, D., D. E. Carling, S. Kuninaga, R. Vilgalys, and M. A. Cubeta. 2001. Ribosomal DNA systematics of Ceratobasidium and Thanatephorus with Rhizoctonia anamorphs. Mycologia 93 (6):1138-1150. Huber, Don M., and Donald R. Sumner. 1996. Suppressive soil amendments for the control of Rhizoctonia species. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. JabajiHare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Hyakumachi, M., and T. Ui. 1987. Non-self-anastomosing isolates of Rhizoctonia solani obtained from fields of sugar-beet monoculture. Transactions of the British Mycological Society 89:155-159. 14 Hyakumachi, M., A. Priyatmojo, M. Kubota, and H. Fukui. 2005. New anastomosis groups, AG-T and AG-U, of binucleate Rhizoctonia spp. causing root and stem rot of cut-flower and miniature roses. Phytopathology 95 (7):784-792. Hyakumachi, M., T. Mushika, Y. Ogiso, T. Toda, K. Kageyama, and T. Tsuge. 1998. Characterization of a new cultural type (LP) of Rhizoctonia solani AG2-2 isolated from warm-season turfgrasses, and its genetic differentiation from other cultural types. Plant Pathology (Oxford) 47 (1):1-9. Ithurrart, M. E. F., G. Buttner, and J. Petersen. 2004. Rhizoctonia root rot in sugar beet (Beta vulgaris ssp altissima) - Epidemiological aspects in relation to maize (Zea mays) as a host plant. Zeitschrift Fur Pflanzenkrankheiten Und Pflanzenschutz-Journal of Plant Diseases and Protection 111 (3):302-312. Johanson, Andrea, Helen C. Turner, Gareth J. McKay, and Averil E. Brown. 1998. A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. FEMS Microbiology Letters 162 (2):289-294. Johnk, Janell Stevens, and Roger K. Jones. 2001. Differentiation of three homogeneous groups of Rhizoctonia solani anastomosis group 4 by analysis of fatty acids. Phytopathology 91 (9):821-830. Katan, Jaacov. 1996. Soil solarization for the control of diseases caused by Rhizoctonia spp. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. Jabaji-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Kataria, H. R., and U. Gisi. 1996. Chemical control of Rhizoctonia species. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Dordrecht / Boston / London: Kluwer Academic Publisher. Kuninaga, S., T. Natsuaki, T. Takeuchi, and R. Yokosawa. 1997. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani. Current Genetics 32 (3):237-243. Kuninaga, Shiro, Donald E. Carling, Toru Takeuchi, and Ryozo Yokosawa. 2000. Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3. Journal of General Plant Pathology 66 (1):2-11. Kuramae, E. E., A. L. Buzeto, M. B. Ciampi, and N. L. Souza. 2003. Identification of Rhizoctonia solani AG 1-IB in lettuce, AG 4 HG-I in tomato and melon, and AG 4 HG-III in broccoli and spinach, in Brazil. European Journal of Plant Pathology 109 (4):391-395. 15 Kuramae, E. E., A. L. Buzeto, A. K. Nakatani, and N. L. Souza. 2007. rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil. European Journal of Plant Pathology 119 (4):469-475. Leach, L. D., and R. H. Garber. 1970. Control of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press. Lehtonen, M. J., P. Ahvenniemi, P. S. Wilson, M. German-Kinnari, and J. P. T. Valkonen. 2008. Biological diversity of Rhizoctonia solani (AG-3) in a northern potato-cultivation environment in Finland. Plant Pathology 57 (1):141-151. Li, Wu, and Yan. 1998. Aetiology of Rhizoctonia in sheath blight of maize in Sichuan. Plant Pathology 47 (1):16-21. Manici, L. M., and P. Bonora. 2007. Molecular genetic variability of Italian binucleate Rhizoctonia spp. isolates from strawberry. European Journal of Plant Pathology 118 (1):31-42. Mazzola, M., R. W. Smiley, A. D. Rovira, and R. J. Cook. 1996. Chracterization of Rhizoctonia isolates, disease occurence and management in cereals. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Dordrecht / Boston / London: Kluwer Academic Publisher. McNeill, J., F. R. Barrie, H. M. Burdet, V. Demoulin, D. L. Hawksworth, K. Marhold, D. H. Nicolson, J. Prado, P. C. Silva, J. E. Skog, J. H. Wiersema, and N. J. Turland. 2006. International Code of Botanical Nomenclature (Vienna Code) adopted by the Seventeenth International Botanical Congress. In International Botanical Congress. Vienna, Austria: International Association for Plant Taxonomy Moore, Royall T. 1987. The Genera of Rhizoctonia-like fungi: Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis, and Rhizoctonia. Mycotaxon 29:91-99. Moore, Royall T. 1996. The dolipore/parenthesome septum in modern taxonomy. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Naito, S. 1996. Basidiospore dispersal and survival. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited 16 by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Naito, Shigeo, and Seiji Kanematsu. 1994. Characterization and pathogenicity of a new anastomosis subgroup AG-2-3 of Rhizoctonia solani Kuhn isolated from leaves of soybean. Annals of the Phytopathological Society of Japan 60 (6):681-690. Ogoshi, A. 1987. Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. In Annual Review of Phytopathology, Vol. 25, edited by R. J. Cook. Palo Alto, California: Annual Reviews Inc. Ogoshi, A. 1996. Introduction - The genus Rhizoctonia. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Ogoshi, A., M. Oniki, T. Araki, and T. Ui. 1983a. Anastomosis groups of binucleate Rhizoctonia in Japan and North America and their perfect states. Nippon Kingakukai Kaiho 24 (1):79-88. Ogoshi, A., Oniki M., Araki T., and Ui T. 1983b. Studies on the anastomosis groups of binucleate Rhizoctonia and their perfect states. Journal of the Faculty of Agriculture Hokkaido University 61 (2):244-260. Olaya, G., and G. S. Abawi. 1991. Occurrence of Thanatephorus cucumeris on table beets in New York state. Phytopathology 81 (10):1186. Olaya, G., and G. S. Abawi. 1994a. Characteristics of Rhizoctonia solani and binucleate Rhizoctonia species causing foliar blight and root rot on table beets in New York state. Plant Disease 78 (8):800-804. Olaya, G., and G. S. Abawi. 1994b. Influence of inoculum type and moisture on development of Rhizoctonia solani on foliage of table beets. Plant Disease 78 (8):805-810. Olaya, G., G. S. Abawi, and J. Barnard. 1994. Response of Rhizoctonia solani and binucleate Rhizoctonia to 5 fungicides and control of pocket rot of table beets with foliar sprays. Plant Disease 78 (11):1033-1037. Oniki, M., A. Ogoshi, T. Araki, R. Sakai, and S. Tanaka. 1985. The perfect state of Rhizoctonia oryzae and Rhizoctonia zeae, and the anastomosis groups of Waitea circinata. Transactions of the Mycological Society of Japan 26 (2):189198. 17 Parmeter, J. R., H. S. Whitney, and W. D. Platt. 1967. Affinities of some Rhizoctonia species that resemble mycelium of Thanatephorus cucumeris. Phytopathology 57 (2):218-223. Pope, E. J., and D. A. Carter. 2001. Phylogenetic placement and host specificity of mycorrhizal isolates belonging to AG-6 and AG-12 in the Rhizoctonia solani species complex. Mycologia 93 (4):712-719. Priyatmojo, Achmadi, Verma E. Escopalao, Naomi G. Tangonan, Cecilia B. Pascual, Haruhisa Suga, Koji Kageyama, and Mitsuro Hyakumachi. 2001. Characterization of a new subgroup of Rhizoctonia solani anastomosis group 1 (AG-1-ID), causal agent of a necrotic leaf spot on coffee. Phytopathology 91 (11):1054-1061. Reiners, S., and C. H. Petzoldt, eds. 2006. 2006 Integrated crop and pest management guidelines for commercial vegetable production. Ithaca: Cornell University Rinehart, T. A., W. E. Copes, T. Toda, and M. A. Cubeta. 2007. Genetic characterization of binucleate Rhizoctonia species causing web blight on Azalea in Mississippi and Alabama. Plant Disease 91 (5):616-623. Roberts, Peter. 1999. Rhizoctonia-forming fungi. Kew: Royal Botanic Gardens. Salazar, Oscar, Maria C. Julian, Mitsuro Hyakumachi, and Victor Rubio. 2000. Phylogenetic grouping of cultural types of Rhizoctonia solani AG 2-2 based on ribosomal ITS sequences. Mycologia 92 (3):505-509. Salazar, Oscar, Johannes H. M. Schneider, Maria C. Julian, Jaap Keijer, and Victor Rubio. 1999. Phylogenetic subgrouping of Rhizoctonia solani AG 2 isolates based on ribosomal ITS sequences. Mycologia 91 (3):459-467. Sharon, Michal, Shiro Kuninaga, Mitsuro Hyakumachi, and Baruch Sneh. 2006. The advancing identification and classification of Rhizoctonia spp. using molecular and biotechnological methods compared with the classical anastomosis grouping. Mycoscience 47 (6):299-316. Sherwood, R. T. 1970. Physiology of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press Sneh, Baruch. 1998. Use of non-pathogenic or hypovirulent fungal strains to protect plants against closely related fungal pathogens. Biotechnology Advances 16 (1):1-32. 18 Sneh, Baruch, L. Burpee, and A. Ogoshi. 1991. Identification of Rhizoctonia species. St. Paul: APS Press. Sneh, Baruch, Suha Jajabi-Hare, Stephen Neate, and Gerda Dijst, eds. 1996. Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control. Boston: Kluwer Academic Publishers. Stalpers, J. A., and T. F. Andersen. 1996. A synopsis of the taxonomy of teleomorphs connected with Rhizoctonia s.l. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. JajabiHare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Stalpers, J. A., T. F. Andersen, and W. Gams. 1998. Two proposals to conserve the names Rhizoctonia and R. solani (Hyphomycetes). Taxon 47 (3):725-726. Sumner, D. R. 1996. Sclerotia formation by Rhizoctonia species and their survival. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Sumner, D. R., and D. K. Bell. 1982a. Crop rotation and yield loss in corn in soil infested with Rhizoctonia solani AG-2 and AG-4. Phytopathology 72 (3):361362. Sumner, D. R., and D. K. Bell. 1982b. Root diseases induced in corn by Rhizoctonia solani and Rhizoctonia zeae. Phytopathology 72 (1):86-91. Toda, T., J. M. Mghalu, A. Priyatmojo, and M. Hyakumachi. 2004. Comparison of sequences for the internal transcribed spacer region in Rhizoctonia solani AG 1-ID and other subgroups of AG 1. Journal of General Plant Pathology 70:270-272. Tomaso-Peterson, M., and L. E. Trevathan. 2004. Rhizoctonia solani AG-13 Isolated from Corn in Mississippi. Tomaso-Peterson, M., and L. E. Trevathan. 2007. Characterization of Rhizoctonia-like fungi isolated from agronomic crops and turfgrasses in Mississippi. Plant Disease 91 (3):260-265. Tu, C. C., and J. W. Kimbrough. 1975. Morphology, development, and cytochemistry of hyphae and sclerotia of species in Rhizoctonia complex. Canadian Journal of Botany-Revue Canadienne De Botanique 53 (20):2282-2296. Tu, C. C., and J. W. Kimbrough. 1978. Systematics and phylogeny of fungi in the Rhizoctonia complex. Botanical Gazette 139 (4):454-466. 19 Tu, C. C., J. W. Kimbrough, and H. C. Aldrich. 1977. Cytology and ultrastructure of Thanatephorus cucumeris and related taxa of Rhizoctonia complex. Canadian Journal of Botany-Revue Canadienne De Botanique 55 (18):2419-2436. Tu, Chin-Chyu, Ting-Fang Hsieh, and Yih-Chang Chang. 1996. Vegetable diseases incited by Rhizoctonia spp. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. JabajiHare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Vilgalys, R., and M. A. Cubeta. 1994. Molecular systematics and population biology of Rhizoctonia. Annual Review of Phytopathology 32:135-155. Win, H. H., and D. R. Sumner. 1988. Root rot induced in snap bean by Rhizoctonia solani AG-4 and AG-2 type 2 in conservation tillage following corn. Plant Disease 72 (12):1049-1053. Yang, C. S., and R. P. Korf. 1985. Ascorhizoctonia new-genus and Complexipes emend., two genera for anamorphs of species assigned to Tricharina discomycetes. Mycotaxon 23:457-482. Yokoyama, K., and A. Ogoshi. 1988. Studies on hyphal anastomosis of Rhizoctonia solani. V. Nutritional conditions for anastomosis. Nippon Kingakukai Kaiho 29 (2):125-132. 20 CHAPTER 2 Characterization of Rhizoctonia solani and Rhizoctonia-like Fungi Infecting Vegetables in New York and their Pathogenicity to Corn Introduction Rhizoctonia solani and Rhizoctonia-like fungi cause root and foliar diseases on various vegetables such as beans, table beets, carrots and cabbage in New York State and other production regions. The damage caused by these fungi in New York on vegetables has increased steadily during the past 10 years. The sexual stage of R. solani, Thanatephorus cucumeris, was first reported in 1990 in New York on table beets (Olaya and Abawi 1991) and may be one of the contributing factors to increased damage by Rhizoctonia-incited diseases through large numbers of aerially dispersed basidiospores (Olaya and Abawi 1994b). Subsequently, T. cucumeris was observed on snap beans and other crops in the state (Abawi et al. 1995). Another factor for the increased prevalence of Rhizoctonia infections was thought to be changes in cultural practices, in which large tractors throw infested soil onto crowns of the plants as they cultivate fields for weed control (Olaya and Abawi 1994b). Control of diseases caused by R. solani and Rhizoctonia-like fungi is difficult due to their soilborne nature, ability to persist as sclerotia, wide host range and versatility (Baker 1970; Katan 1996; Leach and Garber 1970; Menzies 1970; Ogoshi 1996). Today, many growers depend on fungicides to control Rhizoctonia diseases, but they are costly and must be applied at planting or early in the growing season to be effective (Olaya et al. 1994). For vegetable growers, grain crop rotations are recommended and have been effective in suppressing diseases caused by R. solani and Rhizoctonia-like fungi until recently (Abawi and Ludwig 2005; Reiners and Petzoldt 2006). However, during the past few years growers have reported that grain crop 21 rotations have been ineffective (Abawi, personal communication). Increasing disease caused by these fungi and the limited control options available to growers warrant an in depth investigation of this problem. A similar problem has been reported in Germany where high levels of infection on sugar beet has been observed when the crop is in narrow rotation with corn (Ithurrart et al. 2004) Among the anastomosis groups (AGs) of R. solani, isolates belonging to AG 1, AG 2, and AG 4 have been documented to be pathogenic on corn (Garcia et al. 2006; Sneh et al. 1991; Sumner and Bell 1982a, 1982b). In particular, subgroups AG 1-IA and AG 2-2 have been reported to infect corn and cultural type AG 2-2IIIB has been reported to be the causal agent for root rot on corn in the United States (Buddemeyer et al. 2004; Garcia et al. 2006; Ithurrart et al. 2004; Sneh et al. 1991). Previous studies in this region showed that the dominant AGs present in western New York were AG 2-2 on table beets (88%) (Olaya and Abawi 1994a), AG-4 on snap beans (55%) (Galindo et al. 1982), and AG 1 on cabbage (Abawi and Martin 1985). Other isolates found associated with table beets belonged to AG-5, AG-4, AG-2-1, and binucleate Rhizoctonia ( = Ceratobasidium spp.) (Olaya and Abawi 1994a), whereas those from snap beans belonged to AG-1 and AG-2 (Galindo et al. 1982). One explanation of why the corn rotation is ineffective may be the existence of undetected subgroups/cultural types of R. solani that are pathogenic on corn (such as AG 1-IA and AG 2-2IIIB). Previous characterizations of New York isolates of R. solani recovered from vegetables were conducted more than 10 years ago and although AG 1 and AG 2-2 were detected, they were not identified to further subgroups or cultural types. A second potential reason for the ineffectiveness of the corn rotation may be that the isolates that cause disease on vegetables have adapted to infect and/or survive on corn. As an example, AG 5 is commonly found infecting vegetables and turf grasses (Garcia et al. 2006; Sneh et al. 1991), but two recent 22 studies have reported its ability to infect corn (Li et al. 1998; Tomaso-Peterson and Trevathan 2007). In contrast, isolates that usually infect corn may have acquired the ability to infect vegetable crops. For instance, isolates of the Rhizoctonia-like species, Waitea circinata, are generally known to be pathogenic on grasses including corn (Sneh et al. 1991). However, W. circinata var. zeae has been found to cause disease on onions (Erper et al. 2006) and has been isolated from naturally infected soybean and bean in Turkey (Erper et al. 2005). Lastly, an AG or species not known to cause disease on either vegetables or corn may have emerged with the ability to infect these crops. For example, AG-13, usually thought to be non-pathogenic (Garcia et al. 2006), has been isolated from corn in Mississippi (Tomaso-Peterson and Trevathan 2004a). Taxonomic ambiguity among R. solani and Rhizoctonia-like fungi has made studying these fungi challenging (Cubeta and Vilgalys 1997). However, advances in molecular techniques have made characterization of species and AGs easier. Currently, the internal transcribed spacer (ITS) region of the ribosomal DNA (rDNA) is considered most appropriate in characterizing these fungi (Gonzalez et al. 2001; Sharon et al. 2006). DNA sequence data from the ITS region have been used to characterize unknown isolates of R. solani and Rhizoctonia-like fungi to AGs (Kuramae et al. 2007; Lehtonen et al. 2008; Manici and Bonora 2007; Rinehart et al. 2007). To investigate the reasons responsible for the ineffectiveness of corn rotation to suppress Rhizoctonia-incited diseases on vegetables in New York, accurate assessment of the R. solani and Rhizoctonia-like fungal population present in the region is necessary. This study tested three hypotheses that may explain the ineffectiveness of corn rotation in New York: 1. Isolates of R. solani subgroups/cultural type known to be pathogenic to corn, AG 1-IA and AG 2-2IIIB, exist in this region. 2. Isolates of R. solani and Rhizoctonia-like fungi that infect 23 vegetables have gained the ability to infect corn. 3. Non-pathogenic isolates of R. solani and Rhizoctonia-like fungi have emerged with the ability to infect vegetable and corn. These hypotheses were tested by characterizing the R. solani and Rhizoctonia-like fungi infecting vegetables in New York and evaluating corn as a potential host. Methods and Materials Isolate Collection One hundred and fifteen isolates of R. solani and Rhizoctonia-like fungi were recovered from symptomatic vegetable tissues throughout New York State (Table 2.1). Host plants included pea, snap bean, dry bean, cabbage, carrot, and table beet. The fungi were isolated by placing small pieces of infected tissue on acidified water agar medium (pH 3.5). Prior to placement on media, infected tissue pieces were soaked in 10% bleach for 40 seconds to eliminate superficial contaminants and the lesion margins were cut off using a sterile scalpel. Each isolate was established by making a hyphal tip transfer from the margin of a colony exhibiting typical colony morphology and hyphal branching patterns of Rhizoctonia under a dissecting microscope. Isolates were stored in vials on Potato Dextrose Agar (PDA) covered with mineral oil at -4 ºC. Sixty-eight isolates were chosen to represent Rhizoctonia and Rhizoctonia-like fungi causing disease on vegetables in New York for molecular characterization and pathogenicity trials (Table 2.1). 24 Table 2.1 Rhizoctonia solani and Rhizoctonia-like fungi of New York characterized in this study. Isolate number, host plant, location, and date of isolation are listed. Footnotes indicate the rotation crop that the field the isolate was recovered from was planted to in 2005. 25 26 Isolate R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13b R14b R15 R16 R17 R18 R20 R21 R22 R25 R27 R29 R31 R32 R33 R35 R36 R37 R39 R41 R43 R47 Host Plant snap bean snap bean carrot cabbage pea pea pea table beet carrot snap bean snap bean snap bean snap bean snap bean carrot carrot carrot carrot snap bean snap bean snap bean snap bean table beet table beet table beet table beet table beet dry bean dry bean dry bean table beet table beet snap bean table beet Locationa Ontario Ontario Yates Seneca Ontario Ontario Genesee Genesee Unknown (CNY) Livingston Livingston Genesee Genesee Genesee Orleans Orleans Orleans Orleans Orleans Orleans Orleans Orleans Livingston Livingston Livingston Livingston Livingston Livingston Livingston Livingston Livingston Livingston Chemung Genesee Date of collection Fall 2005 Fall 2005 June 2006 June 2006 June 2006 June 2006 June 2006 August 2004 June 2004 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 June 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 Isolate R55 R57 R59 R60 R62 R64 R65 R66c R68c R70c R75 R77 R81 R82 R83 R84 R85 R88 R89 R90 R92 R93 R94 R100 R101 R104 R105 R106 R107 R108 R110 R112 R113 R115 Host Plant snap bean snap bean snap bean snap bean table beet snap bean snap bean table beet table beet table beet snap bean snap bean table beet table beet table beet table beet table beet table beet table beet table beet snap bean snap bean snap bean snap bean snap bean cabbage snap bean carrot cabbage cabbage table beet table beet table beet carrot Locationa Genesee Genesee Ontario Ontario Ontario Genesee Genesee Genesee Genesee Genesee Ontario Ontario Livingston Livingston Livingston Genesee Genesee Genesee Genesee Genesee Genesee Genesee Genesee Genesee Genesee Ontario Ontario Orleans Orleans Orleans Genesee Genesee Genesee Yates Date of collection July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 July 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 July 2006 August 2006 August 2006 August 2006 August 2006 August 2006 August 2006 September 2006 a) Refers to the New York county where the vegetable field was located. (CNY = Central New York) b) Isolate was recovered from a field planted to table beet in 2005. c) Isolate was recovered from a field planted to corn in 2005. 26 Molecular Characterization Individual isolates were grown on PDA at 27 °C and DNA was extracted using the UltracleanTM Soil DNA Isolation Kit (Mo Bio Laboratories, Inc., Carlsbad, CA). The rDNA ITS region consisting of ITS 1, 5.8S, and ITS 2 was amplified using the polymerase chain reaction (PCR) with primers ITS4 and ITS5 for 46 isolates and ITS1 and ITS4 for 22 isolates (Table 2.2) (White et al. 1990). Reactions for PCR amplifications were performed in a 50 µL mixture containing 50 – 100 ng of template DNA, 0.2 µM of each primer, 0.2 mM of each of the four dNTPs, 1.5 units of Taq DNA Polymerase (New England BioLabs, Inc., Ipswich, MA), and 1x ThermoPol Buffer containing 10 mM KCl, 20 mM Tris-HCl, and 2 mM MgSO4 (New England BioLabs, Inc.). The amplifications were performed with a PTC-100TM Peltier Thermal Cycler (MJ Research Inc., Waltham, MA). Cycle parameters were an initial denaturation at 94 °C for 5 min, followed by 35 cycles consisting of denaturation at 94 °C for 1 min, annealing at 56 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. A 4 µL aliquot of each PCR product was run electrophoretically on a 1% agarose gel at 100V to confirm amplification. PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA). The ITS region was sequenced at the Life Sciences Core Laboratories Center of Cornell University using Big Dye Terminator chemistry and the AmpliTaq-FS DNA Polymerase on the Automated 3730 DNA Analyzer (Table 2.2) (Applied Biosystems, Foster City, CA). Sequencing results of many isolates exhibited overlapping peaks in the fluorescent peak trace chromatograms of DNA sequence data. For such isolates PCR products were cloned using the TOPO® TA Cloning Kit for Sequencing with One Shot® TOP10 Chemically Competent E. coli (InvitrogenTM, Carlsbad, CA). Insertion of the ITS region was confirmed by whole-cell PCR. Reactions for PCR 27 Table 2.2 Polymerase chain reaction (PCR) primer combinations, cloning status (Y = cloned, N = not cloned), sequencing primer(s), and GenBank accession numbers for individual isolates used in the molecular characterization. 28 29 Isolate R1 R2 R3 R4 R5 R6 R7 R8 R9 R10 R11 R12 R13 R14 R15 R16 R17 R18 R20 R21 R22 R25 R27 R29 R31 R32 R33 R35 R36 R37 R39 R41 R43 R47 PCR primers ITS4 - ITS5 ITS1 - ITS4 ITS1 - ITS4 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS1 - ITS4 ITS1 - ITS4 ITS1 - ITS4 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS1 - ITS4 ITS1 - ITS4 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 Cloned Y N N N Y Y N Y N N N N Y N Y Y Y Y Y N N Y Y Y N N N N Y N Y Y Y Y Sequencing primers ITS4, ITS5 ITS1 ITS1 ITS4 ITS4, ITS5 ITS4, ITS5 ITS1 ITS4, ITS5 ITS1 ITS1 ITS1 ITS1 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS1 ITS1 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS1 ITS4 ITS1 ITS1 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 GenBank accession No. EU591747 NA EU591748 EU591749 EU591750 EU591751 EU591752 EU591753 EU591754 EU591755 EU591756 EU591757 EU591758 EU591759 EU591760 EU591761 EU591762 EU591763 EU591764 EU591765 EU591766 EU591767 EU591768 EU591769 EU591770 EU591771 EU591772 EU591773 EU591774 EU591775 EU591776 EU591777 EU591778 EU591779 Isolate R55 R57 R59 R60 R62 R64 R65 R66 R68 R70 R75 R77 R81 R82 R83 R84 R85 R88 R89 R90 R92 R93 R94 R100 R101 R104 R105 R106 R107 R108 R110 R112 R113 R115 PCR primers ITS1 - ITS4 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 ITS1 - ITS4 ITS4 - ITS5 ITS1 - ITS4 ITS1 - ITS4 ITS4 - ITS5 ITS4 - ITS5 ITS4 - ITS5 Cloned N Y N N N Y Y Y Y Y Y N Y N Y Y Y Y Y Y N N Y Y Y Y Y N Y N N Y N Y Sequencing primers ITS4 ITS4, ITS5 ITS4 ITS4 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 ITS4 ITS4, ITS5 ITS4 ITS4 ITS4, ITS5 ITS4, ITS5 ITS4, ITS5 GenBank accession No. EU591780 EU591781 EU591782 NA EU591783 EU591784 EU591785 EU591786 EU591787 EU591788 EU591789 EU591790 EU591791 EU591792 EU591793 EU591794 EU591795 EU591796 EU591797 EU591798 EU591799 EU591800 EU591801 EU591802 EU591803 EU591804 EU591805 EU591806 EU591807 EU591808 EU591809 EU591810 EU591811 EU591812 29 amplifications were performed in a 25 µL mixture containing a toothpick scrape of overnight culture, 0.2 µM of each primer, 0.2 mM of each of the four dNTPs, 0.75 units of Taq DNA Polymerase (New England BioLabs, Inc.), and 1x ThermoPol Reaction Buffer containing 10 mM KCl, 20 mM Tris-HCl, and 2 mM MgSO4 (New England BioLabs, Inc.). The amplifications were performed with the PTC-100TM Peltier Thermal Cycler (MJ Research Inc.). Cycle parameters were an initial denaturation at 95 °C for 4 min, followed by 35 cycles consisting of denaturation at 94 °C for 40 sec, annealing at 60 °C for 30 sec, extension at 72 °C for 60 sec, and a final extension at 72 °C for 10 min. A 4 µL aliquot of each PCR product was run electrophoretically on a 1% agarose gel at 100V. For each isolate, one plasmid with confirmed insertion was grown overnight in LB broth medium containing ampicillin and purified using the Wizard® Plus Minipreps DNA Purification System (Promega, Madison, WI). DNA concentration was quantified using the ND-1000 Spectrophotometer (Nanodrop Technologies LLC, Wilmington, DE) and sent to the sequencing facility at Cornell University. The clones were sequenced using both ITS4 and ITS5 primers (Table 2.2) (White et al. 1990). Phylogenetic Analysis Sequence data of complementary strands were checked and edited using SeqEd (Applied Biosystems, Foster City, CA). To infer species, AGs, and subgroups; reference sequences and the outgroup sequence (Athelia rolfsii) from the study conducted by Sharon et al. (2006) were obtained from GenBank (Table 2.3). Additional sequences for AG 13, AG BI, and Ceratobasidium AGs, CAG 1 through CAG 7, were added (Table 2.3) (Carling et al. 2002c; Gonzalez et al. 2001). A reference sequence for CAG 2 was not found, therefore a sequence from AG A (Gonzalez et al. 2001) was used to represent CAG 2, because the two groups are 30 Table 2.3 Reference sequences used in this study to characterize Rhizoctonia solani and Rhizoctonia-like fungi isolated in New York. GenBank accession numbers of the internal transcribed spacer (ITS) sequence, their designated anastomosis group (AG)/subgroup/species, origin, and references are listed (Carling et al. 2002a; Carling et al. 2002c; Ciampi et al. 2005; Godoy-Lutz et al. 2003; Gonzalez et al. 2001; Johanson et al. 1998; Kuninaga et al. 1997; Kuninaga et al. 2000; Kuramae et al. 2003; Pope and Carter 2001; Salazar et al. 2000; Salazar et al. 1999; Sharon et al. 2006; Sharon et al. 2007; Toda et al. 2004; Toda et al. 2007). 31 Species/AG/ subgroup AG 1-IA AG 1-IB AG 1-IC AG 1-ID AG 2-1 AG 2-1-2t AG 2-2LP Rhizoctonia solani AG 2-2IIIB AG 2-2IV AG 2-3 AG 2-4 AG 2-BI AG 3TB AG 3PT AG 4-HGI AG 4-HGII AG 4-HGIII AG 5 AG 6-HGI AG 6-GV1 AG 6-GV2 AG 6-GV3 GenBank accession no. AY270010 AB122133 AB122139 AF308626 AB122142 U19951 AB122128 AB122130 U57729 AY154317 AB054850 AB054852 AB054866 AJ238163 AJ238160 AF354116 AJ238166 AB000014 AY270014 U57740 AB054871 AB054878 AB054879 AB054880 AB054873 AB054875 AF153774 AB000004 AB019023 AB019017 AB000007 AY152704 AB000006 AY154308 AY154659 DQ102449 AF153778 AF354113 AB000019 AF354102 AF153780 AF153782 AY154304 AF354104 AF153788 AF153790 Origin Soybean, Brazil Unknown Unknown Bean, Dominican Republic Unknown Unknown, France Unknown Unknown Pinus, Canada Unknown Tulip, Netherlands Tulip, Netherlands Zoysia grass, Japan Zoysia grass, Japan Zoysia grass, Japan Matrush, Japan Maize, Japan Sugar beet, Japan Unknown Soybean, Japan Soybean, Japan Maize, USA Carrot, USA Carrot, USA Soil, Japan Soil, Japan Tabacco, USA Tabacco, USA Soil, Australia Potato, USA Spinach, Japan Tomato, Brazil Sugar beet, Japan Unknown Spinach, Brazil Soil, Isreal Unknown Sugar beet, Japan Soil, Japan Soil, Japan Unknown, Japan Soil, Japan Unknown Soil, Japan Wheat, South Africa Wheat, Tanzania Reference Ciampi et al., 2005 Toda et al., 2004 Toda et al., 2004 Godoy-Lutz et al., 2003 Toda et al., 2004 Salazar et al., 1999 Toda et al., 2004 Toda et al., 2004 Salazar et al., 1999 Kuramae et al., 2003 Carling et al., 2002b Carling et al., 2002b Carling et al., 2002b Salazar et al., 2000 Salazar et al., 2000 Gonzalez et al., 2001 Salazar et al., 2000 Kuninaga et al., 1997 Ciampi et al., 2005* Salazar et al., 1999 Carling et al, 2002b Carling et al., 2002b Carling et al., 2002b Carling et al., 2002b Carling et al., 2002b Carling et al., 2002b Pope and Carter, 2001 Kuninaga et al., 1997 Kuninaga et al., 2000 Kuninaga et al., 2000 Kuninaga et al., 1997 Kuramae et al., 2003 Kuninaga et al., 1997 Kuramae et al., 2003 Kuramae et al., 2003 Sharon et al., 2007 Pope and Carter, 2001 Gonzalez et al., 2001 Kuninaga et al., 1997 Gonzalez et al., 2001 Pope and Carter, 2001 Pope and Carter, 2001 Kuramae et al., 2003 Gonzalez et al., 2001 Pope and Carter, 2001 Pope and Carter, 2001 32 Table 2.3 continued Species/AG/ subgroup AG 6-GV4 AG 7 AG 8 Rhizoctonia solani AG 9 AG 10 AG 11 AG 12 AG 13 AG BI Waitea circinata Ceratobasidium spp. Athelia rolfsii W. circinata var. zeae W. circinata var. agrostis W. circinata var. circinata W. circinata var. oryzae CAG 1 AG A (CAG 2) CAG 3 CAG 4 CAG 5 CAG 6 CAG 7 A. rolfsii GenBank accession no. AF153785 AF153787 AF354100 AB000003 AF354068 AF153797 AB000011 AF354108 AF354065 AF354071 AF153800 AF153802 AY154313 AF153804 AF153805 AB275645 AB275642 AF354110 AB000044 AB213594 AB213597 AB213575 AB213577 AB213582 AB213581 AJ000195 AB213589 AF354086 AF354092 AF354080 AF354081 AF354082 AF354083 AF354084 AY684917 Origin Leaf litter, Australia Leaf litter, Australia Soil, USA Soil, Japan Barley, Australia Soil, Australia Wheat, Australia Potato, USA Potato, USA Barley, Australia Soil, Australia Lupine, Australia Unknown Pterostylis acuminata Pterostylis acuminata Cotton, USA Cotton, USA Soil, Japan Soil, Japan Soil, Japan Unknown Creeping bentgrass, Japan Kentucky bluegrass, Japan Creeping bentgrass, Japan Unknown Rice, Japan Rice, Japan Turfgrass, USA Soil, Japan Peanut, USA Soybean, USA Cucumber, USA Erigeron, USA Pittosporum, USA Unknown Reference Pope and Carter, 2001 Pope and Carter, 2001 Gonzalez et al., 2001 Kuninaga et al., 1997 Gonzalez et al., 2001 Pope and Carter, 2001 Kuninaga et al., 1997 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Pope and Carter, 2001 Pope and Carter, 2001 Kuramae et al., 2003 Pope and Carter, 2001 Pope and Carter, 2001 Carling et al., 2002a Carling et al., 2002a Gonzalez et al., 2001 Kuninaga et al., 1997 Toda et al., 2007 Toda et al., 2007 Toda et al., 2007 Toda et al., 2007 Toda et al., 2007 Toda et al., 2007 Johanson et al., 1998 Toda et al., 2007 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Gonzalez et al., 2001 Unpublished * The GenBank accession number is not listed in the publication but inferred by GenBank. 33 known to be equivalent between the Japanese and American AGs (Table 2.3) (Garcia et al. 2006). Sequences of collected isolates and reference sequences were aligned using Clustal X (1.81) (Thompson et al. 1997) and its default parameters. Sequence alignment was adjusted manually by visual examination using MacClade 4.08 (Maddison and Maddison 2005). The sequence data set was analyzed using two analyses, neighbor-joining and Bayesian inference. For neighbor-joining analysis, the appropriate distance model was chosen according to the guidelines described in the MEGA version 4 manual (Tamura et al. 2007) based on calculating pair-wise distances (d) using the JukesCantor distance model (Jukes and Cantor 1969) and the transition/transversion ratio (R). Since d < 0.3 (0.00 < d < 0.272) and R was low (R = 1.164), the Jukes-Cantor distance model (Jukes and Cantor 1969) was selected. A neighbor-joining tree (Saitou and Nei 1987) was generated using PAUP* ver. 4.0b10 (Swofford 2004) with bootstrap values based on 1000 replicates. For Bayesian inference, the HasegawaKishino-Yano plus Gamma (HKY+G) model (Hasegawa et al. 1985) was chosen as the appropriate evolutionary model by Modeltest 3.7 (Posada and Crandall 1998) using the Akaike Information Criterion (Akaike 1974). The output parameters (number of substitution types = 2, among-site rate variation = gamma, trasition/transversion rate ratio = 1.27, state frequencies (A, C, G, T) = (0.27, 0.21, 0.16, 0.36), proportion of invariable sites = 0, gamma shape = 0.35) were entered into MrBayes 3.2.1 (Ronquist and Huelsenbeck 2003). Two million generations were run and trees were sampled every 100 generations. The first 2000 trees were discarded as the burn-in and a strict consensus tree was generated. Both neighbor-joining and Bayesian inference analyses included 833 characters and were unrooted. A. rolfsii was positioned as the outgroup after constructing the trees. 34 Pathogenicity Evaluation on Corn in the Greenhouse To determine whether the representative isolates from New York were capable of causing disease on corn, a series of greenhouse bioassays were performed. Untreated sweet white corn cultivar Silver Princess was planted in 3.8 x 21 cm conetubes (Stuewe & Sons, Inc). Approximately 200 cm3 of pasteurized soil (60 °C for 30 min) was placed in each tube and planted with two seeds of corn. Inoculum of selected collected isolates was prepared by growing isolates on PDA for 5-7 days. Seven or eight days after planting, the soil adjacent to the stem of corn seedlings was removed and a colonized PDA disk (8 mm in diameter) was placed in the pocket against the lower stem (Figure 2.1). Non-colonized PDA disks were placed next to the stems of the negative controls. After inoculation, the disks were covered with soil to prevent drying. An individual tube represents a replication and each trial consisted of four replications per isolate. Inoculation trials were conducted at four time-points; two trials were conducted for each isolate on different dates and all four trials included negative controls; thus with a total of 16 observations for each isolate and 32 observations for the negative control. After two weeks of incubation (Figure 2.2), plants were carefully removed from the soil and the roots were washed. Disease severity was assessed using a rating scale from 0 to 5; where 0 = no symptoms observed (healthy), 1 = lesions smaller than 2 mm, 2 = lesions larger than 2 mm, 3 = girdling lesion on crown tissue, 4 = rotted mesocotyl causing wire-stem symptoms, and 5 = dead seedling. Examples of symptoms for the various categories are shown in Figure 2.3. After harvest, pieces of symptomatic corn tissue from several treatments were placed on acidified PDA to ascertain the presence and recovery of the same R. solani or Rhizoctonia-like fungi used in the inoculation. 35 Figure 2.1 Method of inoculation of corn seedlings: One week after planting, soil adjacent to the lower stem was removed and colonized potato dextrose agar (PDA) disks were place in the pocket. The disks were then covered with soil to prevent drying. 36 Figure 2.2 Incubation of corn seedlings in the greenhouse for 2 weeks after inoculation. 37 0 23 4 Figure 2.3 Representation of the disease rating scale (0 to 5) used to assess disease severity on corn caused by Rhizoctonia solani and Rhizoctonia-like fungi tested in this study: 0 = no symptoms, 1 = lesions smaller than 2 mm, 2 = lesions larger than 2 mm, 3 = girdling lesions on crown tissue, 4 = rotted mesocotyl tissue with wire-stem symptoms, and 5 = dead seedlings. Pictures are from corn seedlings that were in the actual experiments. 38 Pathogenicity Evaluation on Snap Beans in the Greenhouse This preliminary experiment was conducted to compare the virulence of the collected R. solani and Rhizoctonia-like isolates on corn to that on vegetables. The snap bean cultivar Hystyle was used as a model for vegetables to contrast the virulence of selected isolates in the greenhouse. Seven isolates (R18, R20, R25, R31, R39, R47, and R62) that showed variable virulence on corn were chosen to inoculate snap bean plants. Seeds of snap bean cultivar Hystyle treated with Captan®, Maxim®, and Thiram® were planted in 10 cm diameter clay pots (4 seeds per pot) filled with pasteurized soil. Inoculum of the collected isolates was prepared by growing the isolates on PDA for 10 days. Eleven days after planting, snap bean seedlings were inoculated in the same way the corn seedlings were inoculated as mentioned above. After inoculation, the disks were covered with soil to prevent drying. An individual pot represented a replication and there were four replications per isolate. The trial was conducted only once. After two weeks of incubation, plants were carefully removed from the soil and the roots and stems were washed. Disease severity was assessed using a rating scale of 0 to 3; where 0 = no symptoms (healthy), 1 = superficial lesions, 2 = sunken distinct lesions, 3 = rotted lower hypocotyl tissues showing initial symptoms of wire-stem. Examples of symptoms for the various disease categories are shown in Figure 2.4. Statistical Analysis Data from both corn and snap bean experiments were analyzed using SAS® software Version 9 (SAS Institute Inc., Cary, NC). The ordinal data did not have a normal distribution, therefore they were analyzed using the non-parametric methodology of Brunner and colleagues (Brunner et al. 2002) as described by (Shah and Madden 2004). For analyses on corn, data were pooled from the two trials to 39 01 23 Figure 2.4 Representation of the disease rating scale (0 to 3) used to assess disease severity on snap beans caused by Rhizoctonia solani and Rhizoctonia-like fungi tested in this study: 0 = no symptoms, 1 = superficial lesions, 2 = sunken distinct lesions, 3 = rotted hypocotyl tissues with initial symptoms of wire-stem. Pictures are from snap bean seedlings that were in the actual experiments. 40 Figure 2.5 A subset of Rhizoctonia solani and Rhizoctonia-like fungi collected in New York to show their morphological diversity. Isolates were grown on PDA for 2 weeks. 41 obtain overall results for analysis on corn. Two analyses were performed; the first analysis assessed disease severity caused by individual isolates and the second analysis assessed disease severity in relation to the inferred identification of isolates. For analysis on snap bean, the incidence of infection on snap bean was relatively low, therefore only infected plants were included in the analysis. Results Phylogenetic analysis In the neighbor-joining tree, collected isolates were inferred to belong to an AG, subgroup, or species if they formed a cluster including a reference sequence supported by a bootstrap value (BS) of 95% or higher, and were considered closely related to a group if supported by a BS lower than 95%. The analysis inferred the identification of 61 isolates: Twenty-six isolates (38%) were AG 2-2, 19 isolates (28 %) were AG 4, 6 isolates (9%) were AG 1, 3 isolates (4%) were AG 2-1, 2 isolates (3%) were AG 5, 2 isolates were W. circinata var. zeae, 1 isolate (1%) was AG 11, 1 isolate (1%) was CAG 2, and 1 (1%) isolate was CAG 6. The remaining 7 isolates were closely related to CAG 2 supported by a BS 87% (Figure 2.6). As for identifying isolates to further subgroup of cultural type, within AG 2-2; 10 isolates belonged to AG 2-2IV, within AG 4; 1 isolate belonged to AG 4-HGI and 15 isolates were closely related to AG 4-HGII (BS 81%), and within AG 1; 4 isolates belonged to AG 1-IC and 2 isolates belonged to AG 1-IB (Figure 2.6). Remaining isolates within AG 2-2 and AG 4 could not be identified to further subgroup or cultural type. In the Bayesian inference tree, collected isolates were inferred to belong to an AG, subgroup, or species if they formed a cluster including a reference sequence supported by a posterior probability value (PP) of 90% or higher and were considered closely related to a group if supported by a PP lower than 90%. Using this criterion, 42 Figure 2.6 Neighbor-joining tree generated from the rDNA ITS1-5.8S- ITS2 region of the nuclear ribosomal DNA of reference sequences and collected New York isolates. Bootstrap values are based on 1000 replicates and values greater than 50% are indicated in italics. Reference sequences are indicated with GenBank accession numbers followed by the species, AG, or subgroup designation and New York isolates are indicated by the isolate number following the letter R. Inferred identifications are indicated on the right (W. c. v. zeae = Waitea circinata var. zeae). Host of origin of collected New York isolates are indicated after the isolate number: CG = cabbage, CT = carrot, DB = dry bean, P = pea, SB = snap bean, TB = table beet. 43 AG 4 AG 1 AG 2-1 100 0.005 substitutions/site 100 AY154659 4 HGIII 56 70 DQ102449 4 HGIII AB000007 4 HGI R82 - TB AY152704 4 HGI R57- SB R64 - SB AY154308 4 HGII R112 - TB ARRRB1914210000--- 0STSBBB06 4 HGII 100 R5 - P 67 R43 - SB 72 R10 - SB 78 R11 - SB R9 - CT R12 - SB 81 R6 - P R94 - SB R101 - SB R106 - CT R55 R65 - SB 100 -US1B9951 1 IC AB122142 1 IC R3- CT R17 - CT R108 - CG 88 R107 - CG 100 AB122139 1 IB 92 R4 - CG R93 - SB AF308626 1 IB 78 100 AB122130 1 ID 100 AB122128 1 ID AY270010 1 IA AB122133 1 IA 67 100 AB275645 13 100 AB275642 13 AF153804 12 82 AF153805 12 AF153790 6 GV3 94 AF153788 6 GV3 AF153785 6 GV4 58 96 AF153787 6 GV4 AY154304 6 GV2 100 AF354104 6 GV2 AF153782 6 GV1 99 AF153780 6 GV1 AF354102 6 HGI AB000019 6 HGI 100 AB054873 2 BI AF354110 BI AB054875 2 BI 98 AB000044 BI 75 AF354071 10 AF153800 10 100 AB054879 2 4 AB054880 2 4 96 AB054878 2 4 AF354065 9 AF354108 9 73 AY154317 2 1 99 AB054852 2 1 2t 99 AB054850 2 1 2t U57729 2 1 R16 - CT 60 R104 - CG 96 100 R15 - CT 91 AF153774 3TB 100 AB000004 3TB AB019023 3PT AB019017 3PT AB000014 2 2IV R85 - TB R31 - TB R33 - TB R83 - TB R90 - TB 95 R81 - TB R32 - TB R88 - TB R84 - TB 91 R89 - TB AY270014 2 2IV AJ238164 2 2IV 97 AJ238160 2 2LP AJ238163 2 2LP 99 AB054866 2 2LP R70 - TB R115 - CT 100 R105 - SB R68 - TB R27 - TB 90 100 R39- - TB CT R2 - SB RR71500- SB R47 - TB R1 - TB AJ238166 2 2IIIB 100 AF354116 2 2IIIB R8 - TB R29 - TB 96 R25 - SB R66 - TB 53 100 R41 - TB AY154313 11 78 96 U57740 R22 23 - SB 53 AB054871 2 3 AF153802 11 100 AF153778 5 AF354113 5 R35 - DB R7 - P 79 AB000011 8 AF354068 8 AF153797 8 100 AF354100 7 97 AB000003 7 AF354083 CAG6 65 R77 - SB AF354082 CAG5 AF354080 CAG3 AF354084 CAG7 99 AF354081 CAG4 AF354092 A CAG2 61 R21 - SB R37 - SB R62 - TB 87 R113 - TB R36 - DB R60- SB R20- SB R59- SB AF354086 CAG1 98 AB213594 ZEAE 85 R18 - CT AB213597 ZEAE 100 R13 - SB AB213575 AGROSTIS 100 AB213577 AGROSTIS AB213589 ORYZAE 100 AJ000195 ORYZAE AB213582 CIRCINATA AB213581 CIRCINATA AY684917 AR AG 2-2 AG 11 AG 5 CAG 6 CAG 2 W. c. v. zeae 44 Bayesian analysis (Figure 2.7) supported the inferences suggested by the neighborjoining analysis for 61 isolates. As for the remaining 7 isolates; 5 isolates were closely related to CAG 2 (PP 38%) and the identity of 2 isolates (R20 and R59) could not be determined. In comparison to the neighbor-joining analysis, the Bayesian tree was better resolved and permitted tentative inferences about subgroups and cultural types. Within AG 2-2; 5 isolates belonged to AG 2-2IIIB, 1 isolate was closely related to AG 2-2IIIB (PP 86%), 16 isolates were closely related to AG 2-2IV (PP 80% and PP 64%), and 4 isolates were closely related to AG 2-2LP (PP 84%). Within AG 4; 1 isolate belonged to AG 4-HGI, 1 isolate was closely related to AG 4-HGI (PP 77%), 15 isolates belonged to AG 4-HGII, and 1 isolate was closely related to AG 4-HGII (PP 58%). Within AG 1; 2 isolates belonged to AG 1-IB and 4 isolates belonged to AG 1-IC (Figure 2.7). Pathogenicity Evaluation on Corn in the Greenhouse Disease severity on corn caused by R. solani and Rhizoctonia-like fungi isolated in New York was evaluated using relative treatment effects and their 95% confidence intervals (CI) (Table 2.4). Among the 32 observations of the negative control, 2 plants were minimally infected with a disease rating of 1. This was potentially due to contamination through splashing of soil during watering. Sixty-one isolates were considered pathogenic to corn, whereas pathogenicity of the remaining 7 isolates (R20, R62, R60, R65, R18, R59, and R36) could not be confirmed as it was within the range of the chance of contamination. There were significant differences in disease severity on corn caused by individual isolates (Table 2.4). In addition, there were significant differences in disease severity on corn when the isolates were grouped according to inferred AG/subgroup/species of R. solani and Rhizoctonia-like 45 Figure 2.7 Bayesian inference tree generated from the rDNA ITS1-5.8S- ITS2 region of the nuclear ribosomal DNA of reference sequences and collected isolates. Bayesian posterior probability values greater than 50% are indicated in italics. Reference sequences are indicated with GenBank accession numbers followed by the species, AG, or subgroup designation and New York isolates are indicated by the isolate number following the letter R. Inferred identifications are indicated on the right (W. c. v. zeae = Waitea circinata var. zeae). Host of origin of collected New York isolates are indicated after the isolate number: CG = cabbage, CT = carrot, DB = dry bean, P = pea, SB = snap bean, TB = table beet. 46 100 92 100 AY684917 AR 88 AB213594 ZEAE 100 R18 - CT 73 100 AB213597 ZEAE R13 - SB 100 AB213575 AGROSTIS 100 AB213577 AGROSTIS 73 AB213589 ORYZAE AJ000195 ORYZAE AB213582 CIRCINATA AB213581 CIRCINATA 10 changes 100 R20 - SB R59 - SB 96 AF354092 A CAG2 R21 - SB 55 R37 - DB 55 R113 - TB R60 - SB R36 - DB R62 - TB 47 100 AY154659 4 HGIII DQ102449 4 HGIII AB000007 4 HGI 77 99 R82 - TB AY152704 4 HGI R57 - SB R64 - SB AY154308 4 HGII R43 - SB R106 - CT 100 95 R112 - TB R92 - SB R55 - SB R14 - SB R10 - SB R11 - SB 70 R6 R110 -P - TB R94 - SB R5 R9 -P R12 - CT AB000-0S0B6 4 HGII R101 - SB R65 - SB U19951 1 IC R17 - CT 100 R108 - CG 92 57 R3 - CT RAB1017221-4C2G 1 IC 100 AY270010 1 IA AB122133 1 IA 91 AB122139 1 IB 100 R4 - CG R93 - SB 78 87 AF308626 1 IB AB122130 1 ID AB122128 1 ID 99 AB275645 13 AB275642 13 AF153804 12 AF153805 12 AF153790 6 GV3 AF153788 6 GV3 AF153785 6 GV4 AF153787 6 GV4 AY154304 6 GV2 AF354104 6 GV2 AF153782 6 GV1 AF153780 6 GV1 AF354102 6 HGI AB000019 6 HGI 99 100 AF354083 CAG6 99 R77 - SB AF354081 CAG4 AF354082 CAG5 AF354080 CAG3 AF354084 CAG7 AB054873 2 BI 100 AF354110 BI AB054875 2 BI 71 AB000044 BI AF354065 9 AF354108 9 AY154317 2 1 AB054852 2 1 2t 100 AB054850 2 1 2t U57729 2 1 R16 - CT R104 - CG R15 - CT 96 AF354071 10 AF153800 10 AB054879 2 4 AB054878 2 4 AB054880 2 4 AF153774 3TB 100 AB000004 3TB AB019023 3PT AB019017 3PT AB000014 2 2IV 84 R31 - TB R33 - TB R32 - TB R85 - TB 80 R90 - TB R83 - TB R81 - TB R88 - TB R84 - TB R89 - TB AY270014 2 2IV 100 AJ238164 2 2IV 64 R2 - SB R100 - SB R39 - TB R75 - SB R1 - SB R47 - TB AJ238160 2 2LP AJ238163 2 2LP AB054866 2 2LP R70 - TB R115 - CT R105 - SB R68 - TB R27 - TB AJ238166 2 2IIIB AF35-4T1B16 2 2IIIB 94 R8 - TB 98 R41 R25 - SB R66 - TB R29 - TB 100 AY154313 11 R22 - SB U57740 2 3 100 AB054871 2 3 AF153802 11 100 AF153778 5 AF354113 5 R35 - DB R7 - P 99 AB000011 8 AF153797 8 AF354068 8 100 AF354100 7 AB000003 7 AF354086 CAG1 CAG 2 W. c. v. zeae AG 11 AG 5 AG 2-2 AG 2-1 CAG 6 AG 1 AG 4 Table 2.4 Median and relative treatment effects (p) with 95% confidence intervals (CI) of disease severity ratings obtained for individual isolates from the corn and snap bean pathogenicity trials. Isolates are ranked in ascending order of relative treatment effects from the corn pathogenicity trial. Each isolate is accompanied by the inferred identification of anasotmosis group (AG), subgroup, or species and the host of isolation. Disease severity ratings on corn were determined on a scale of 0 to 5, and those on snap bean were determined on a scale of 0 to 3. Relative treatment effects were calculated using the non-parametric method for ordinal data described by Shah and Madden (2004). 48 Isolate Negative control R20 R62 R60 R65 R18 R59 R36 R37 R15 R16 R9 R77 R113 R14 R104 R12 R112 R94 R21 R110 R5 R55 R92 R107 R108 R105 R115 R6 R17 R106 R43 R83 R4 R13 Inferred AG NA CAG 2 CAG 2 CAG 2 AG 4 W. circinata var. zeae CAG 2 CAG 2 CAG 2 AG 2-1 AG 2-1 AG 4 CAG 6 CAG 2 AG 4 AG 2-1 AG 4 AG 4 AG 4 CAG 2 AG 4 AG 4 AG 4 AG 4 AG 1 AG 1 AG 2-2 AG 2-2 AG 4 AG 1 AG 4 AG 4 AG 2-2 AG 1 W. circinata var. zeae Host Plant NA snap bean table beet snap bean snap bean carrot snap bean dry bean dry bean carrot carrot carrot snap bean table beet snap bean cabbage snap bean table beet snap bean snap bean table beet pea snap bean snap bean cabbage cabbage snap bean carrot pea carrot carrot snap bean table beet cabbage snap bean Median 0 0 0 0 0 0 0 0 0 0 0 0.5 0.5 0.5 1 0.5 1 0.5 0 1 1 1 1.5 1 2 2 2 1 1.5 2 2 2 1.5 2 2 Corn p 0.151 0.166 0.191 0.207 0.207 0.22 0.22 0.26 0.26 0.273 0.291 0.301 0.301 0.308 0.312 0.316 0.329 0.332 0.344 0.372 0.39 0.393 0.413 0.424 0.452 0.452 0.462 0.468 0.477 0.478 0.478 0.484 0.491 0.525 0.54 95% CI for p (0.131, 0.17) (0.111, 0.221) (0.087, 0.295) (0.131, 0.283) (0.131, 0.283) (0.142, 0.297) (0.142, 0.297) (0.163, 0.357) (0.173, 0.348) (0.187, 0.36) (0.188, 0.393) (0.209, 0.394) (0.209, 0.394) (0.197, 0.418) (0.231, 0.393) (0.218, 0.415) (0.233, 0.425) (0.228, 0.435) (0.213, 0.474) (0.271, 0.473) (0.257, 0.523) (0.282, 0.503) (0.319, 0.507) (0.345, 0.503) (0.334, 0.571) (0.334, 0.571) (0.328, 0.597) (0.33, 0.605) (0.381, 0.573) (0.372, 0.584) (0.372, 0.584) (0.403, 0.565) (0.33, 0.651) (0.471, 0.579) (0.443, 0.636) Median 0 1 1 Snap Bean p 0.269 0.516 0.487 95% CI for p (0.228, 0.31) (0.401, 0.631) (0.376, 0.599) 1 0.419 (0.313, 0.525) 49 49 Table 2.4 continued Isolate R101 R35 R68 R3 R82 R90 R31 R22 R70 R10 R32 R93 R57 R33 R66 R88 R11 R64 R81 R89 R84 R1 R7 R29 R41 R27 R85 R100 R47 R2 R8 R25 R75 R39 Inferred AG AG 4 AG 5 AG 2-2 AG 1 AG 4 AG 2-2 AG 2-2 AG 11 AG 2-2 AG 4 AG 2-2 AG 1 AG 4 AG 2-2 AG 2-2 AG 2-2 AG 4 AG 4 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 5 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 AG 2-2 Host Plant snap bean dry bean table beet carrot table beet table beet table beet snap bean table beet snap bean table beet snap bean snap bean table beet table beet table beet snap bean snap bean table beet table beet table beet snap bean pea table beet table beet table beet table beet snap bean table beet snap bean table beet snap bean snap bean table beet Median 2 2 2 2 2 2 3 2 2 2 2.5 2 2 2 2 2 2 2 2 2 3 2.5 2 2.5 3 2.5 3 3 3 3 3.5 3 3.5 4 Corn p 0.552 0.553 0.555 0.558 0.564 0.564 0.572 0.574 0.574 0.59 0.593 0.599 0.606 0.617 0.631 0.633 0.64 0.646 0.646 0.654 0.68 0.697 0.697 0.717 0.727 0.732 0.733 0.775 0.777 0.785 0.792 0.813 0.818 0.898 95% CI for p (0.467, 0.638) (0.44, 0.667) (0.415, 0.695) (0.487, 0.628) (0.487, 0.642) (0.43, 0.697) (0.412, 0.732) (0.435, 0.713) (0.438, 0.711) (0.501, 0.679) (0.453, 0.734) (0.49, 0.708) (0.522, 0.689) (0.516, 0.718) (0.496, 0.765) (0.51, 0.755) (0.536, 0.744) (0.558, 0.733) (0.543, 0.748) (0.553, 0.755) (0.551, 0.809) (0.597, 0.798) (0.612, 0.783) (0.608, 0.826) (0.61, 0.844) (0.656, 0.808) (0.653, 0.814) (0.699, 0.851) (0.674, 0.88) (0.694, 0.876) (0.699, 0.884) (0.739, 0.887) (0.735, 0.901) (0.872, 0.924) Median Snap Bean p 95% CI for p 1 0.518 (0.389, 0.647) 1 0.505 (0.379, 0.632) 1 0.518 (0.389, 0.647) 2 0.795 (0.703, 0.886) 50 50 fungi (Figure 2.8). AG 2-2 isolates were the most virulent, whereas CAG 2 isolates were the least virulent on corn (Figure 2.8). Pathogenicity evaluation on snap bean Disease severity on snap bean caused by the selected 7 isolates of R. solani and Rhizoctonia-like fungi collected in New York was evaluated using relative treatment effects and their 95% CI (Table 2.4). All isolates tested were pathogenic to snap bean. Also, there were significant differences in disease severity caused by the selected isolates on snap bean. Isolate R18 was the least virulent on snap beans, whereas isolate R39 was the most virulent (Table 2.4). Discussion Molecular characterization of R. solani and Rhizoctonia-like fungi in New York showed the presence of a diverse population with isolates belonging to AG 1, AG 2-2, AG 4, AG 5, AG 11, CAG 2, CAG 6, W. circinata var. zeae, and several isolates closely related to CAG 2 (Figure 2.6, Figure 2.7). The dominant groups were AG 2-2 (38%) and AG 4 (28%). As most isolates were collected from table beets and snap beans, this finding is consistent with results reported previously from New York (Galindo et al. 1982; Olaya and Abawi 1994a). This is the first report of W. circinata var. zeae isolation from vegetable tissues, specifically from naturally infested carrot and snap bean plants in western New York. There are only two other reports from Turkey where this fungus has been isolated from vegetables (Erper et al. 2005; Erper et al. 2006). It was also the first time that AG 11 (isolate R22) was isolated in this region. AG 11 reported to be pathogenic on wheat and lupine (Carling et al. 1994; Garcia et al. 2006; Sweetingham 1989), but isolate R22 was isolated from snap bean. 51 Relative treatment effects 0.8 0.7 0.6 0.5 0.4 b 0.3 0.2 a 0.1 0 Disease severity c,d d,e d,e d b b,c c b AG2-2 AG5 AG11 AG1 AG4 W.c.v.z. CAG6 AG2-1 CAG2 0 Inferred group Figure 2.8 Disease severity on corn caused by Rhizoctonia solani and Rhizoctonia-like isolates collected in New York, when grouped by inferred anastomosis group (AG), subgroup, or species. Disease severity ratings were determined on a scale of 0 to 5. Relative treatment effects were calculated using the non-parametric method for ordinal data described by Shah and Madden (2004). Group 0 indicates the negative control and group W.c.v.z. indicates W. circinata var. zeae. Bars indicate 95% CI of relative treatment effects. Different letters indicate significant differences between groups using the 95% CI. 52 Isolates from AGs that are documented to be pathogenic on corn, AG 1, AG 2, and AG 4 were present. Within these AGs, we specifically sought the presence of subgroups/cultural types that are well-documented to be pathogenic on corn, AG 1-IA and AG 2-2IIIB, were examined. No isolates of AG 1-IA were detected; however, 6 isolates of AG 2-2IIIB were recovered in this investigation. Thus, with 6 isolates belonging to AG 2-2 IIIB, the hypothesis that subdivisions pathogenic to corn exist in New York cannot be rejected. AG 2-2 is subdivided into cultural types based on pathogenicity on mat rush, sugar beet, and warm season turf grasses: AG 2-2IIIB is designated as the rush type, AG 2-2IV is designated as the root rot type, and AG 22LP is designated as the large patch type (Hyakumachi et al. 1998; Ogoshi 1987). Despite the originally proposed characterization of AG 2-2 subdivisions, our results show that all three cultural types are able to infect corn and the hosts from which they were isolated, primarily snap bean and table beet. This suggests a need for a more thorough characterization criterion for AG 2-2 subdivisions. Among the R. solani and Rhizoctonia-like fungi isolated from vegetables in New York, R. solani AG 2-2 isolates were most virulent on corn, AG 4 isolates were moderately virulent, and binucleate CAG 2 isolates were least virulent (Figure 2.8). These results are in agreement with those reported previously by Ithurrart, Buttner, and Petersen (2004), where an increase in disease on sugar beet was reported to occur when a narrow sugar beet-corn rotation was practiced. Most AG 2-2 isolates in this study were recovered from table beets. This explains why the earliest observations of the ineffectiveness of corn rotation in reducing severity of Rhizoctonia diseases on vegetables in New York were made by beet growers (Abawi, personal communication). Additionally, AG 2-2 has been confirmed to produce the sexual state, T. cucumeris, on table beets in commercial fields in New York (Olaya and Abawi 1994a). This suggests the possibility that AG 2-2 isolates of R. solani are 53 evolving pathogenicity traits to corn through sexual recombination. In combination with the isolation of W. circinata var. zeae from symptomatic vegetables, these findings suggest the ineffective corn rotation may be due to an expansion in host range. AG 2-2 isolates may have acquired the ability to infect corn, while W. circinata var. zeae isolates may have gained the ability to infect vegetables. Further investigation of the host range of these isolates is necessary to confirm this hypothesis. AGs or species previously considered to be non-pathogenic were not recovered in this investigation. Therefore, the emergence of new pathogenic AGs or species does not appear to be the reason behind the ineffectiveness of corn rotation reported by vegetable growers in New York. Our results also revealed differences in AGs recovered from different hosts. Most isolates recovered from table beets belonged to AG 2-2 and most isolates recovered from cabbage belonged to AG 1. However, isolates recovered from carrot and snap bean belonged to many AGs. This suggests that certain hosts are susceptible to specific AGs, whereas other hosts are susceptible to a wide array of AGs (Figure 2.6, Figure 2.7, Table 2.4). The preliminary pathogenicity trial on snap beans suggested a potential correlation between virulence of the collected isolates on corn and snap bean. Isolates R18, R20, R62 exhibited relatively low virulence on corn, whereas isolates R25, R31, R39, and R47 were higher in virulence. Similarly, isolates R18 and R62 were low in virulence on snap bean, whereas isolates R25, R31, and R39 exhibited higher virulence (Table 2.4). Thus, there is a correlation between the virulence on corn and snap bean for these isolates. Due to the small number of isolates that were tested for virulence on snap bean, the correlation between the virulence of the isolates on corn and snap bean should be further tested before definitive conclusions are made. If such correlation between virulence to corn and virulence to snap bean or other vegetables 54 can be proven with a larger number of isolates and under field conditions, it can then be recommended to avoid the use of corn as a rotation crop for snap bean or other vegetables where R. solani and Rhizoctonia-like fungi are prevalent. In both the neighbor-joining and Bayesian trees, reference sequences of AG 2 and CAGs did not group with other reference sequences representing the same group. As for AG 2 subgroups that did not cluster together, similar results have been reported by Kuninaga et al. (1997) suggesting that subgroups within AG 2 may be phylogenetically distant and there are different opinions about whether AG 2 subgroups should be considered as distinct AGs or not (Carling 1996). Unlike other AG subgroups that are determined by pathogenicity, morphology, and biochemical properties, AG 2 subgroups are determined by hyphal fusion frequency (Carling 1996; Ogoshi 1987; Salazar et al. 1999), a criterion similar to that used in characterizing AGs. Taking this into consideration, AG 2 subgroups may be more representative of distinct AGs than subgroups and our analyses support this. As for the CAG reference sequences that did not group together, similar results have been reported by Gonzalez et al. (2001) and the taxonomy of Ceratobasidium and Thanatephorus is still under debate (Gonzalez et al. 2001). A more rigorous, multilocus approach is necessary to clarify the phylogeny of these two genera. Our analyses suggest the use of the rDNA ITS sequence in either neighbor-joining or Bayesian inference to be robust enough to identify isolates to species and AG. However, in order to classify isolates to subgroup or AG 2-2 cultural type, Bayesian inference appears to be more predictive than neighbor-joining (Figure 2.6, Figure 2.7). Sharon et al. (2006) have demonstrated that the rDNA ITS sequence cannot differentiate cultural types of AG 2-2 when used in a neighbor-joining analysis. Developing a simple approach to identify isolates among a diverse population to AG subgroups may be challenging because subgroups have been constructed under independent criteria for different AGs. For example, AG 1 55 subgroups are based on morphology and pathogenicity, AG 2 subgroups are based on hyphal fusion frequencies, while AG 8 subgroups are based on zymogram patterns (Carling 1996). Therefore, implementing a universal criterion to characterize AG subgroups may allow the development of a robust method to identify isolates to subgroups and subdivisions. Our results suggest that R. solani and Rhizoctonia-like fungi that infect vegetables in western New York, especially AG 2-2 isolates, are capable of infecting corn as well. Visual observations of symptoms caused by these isolates and their reisolation from inoculated seedlings clearly indicate that corn serves as a host plant. Isolates R66, R68, and R70 were collected from a field that was in corn rotation during 2005 and isolates R13 and R14 were collected from a field that was in table beet during 2005 (Table 2.4). The corn rotation isolates were characterized as AG 2-2 and R13 and R14 were characterized as W. circinata var. zeae and AG 4, respectively. Disease severity caused by the corn rotation isolates on corn was as high as or higher than that caused by the vegetable isolates. These findings suggest that corn should be avoided as a rotation crop especially when AG 2-2 is present in the field. Following up this study with larger sample sizes from each AG may provide a better understanding of when corn rotations should be avoided due to the presence of certain genotypes of Rhizoctonia in the field. In this study we looked at one sweet corn cultivar, but it will be interesting to see if certain corn cultivars are resistant to infection by R. solani and Rhizoctonia-like fungi from New York. Additionally, in contrast to the controlled environment of a greenhouse, studies should also be conducted in the field to simulate natural conditions. Future investigations to evaluate the effectiveness of other cereals as rotation crops against R. solani and Rhizoctonialike fungi are also needed. 56 REFERENCES Abawi, G. S., and S. B. Martin. 1985. Rhizoctonia foliar blight of cabbage in New York State. Plant Disease 69 (2):158-161. Abawi, G. S., and J. W. Ludwig. 2005. Effect of three crop rotations with and without deep plowing on root rot severity and yield of beans. Annual Report of the Bean Improvement Cooperative 48:118-119. Abawi, G. S., G. Olaya, and J. W. Ludwig. 1995. Occurrence of Thanatephorus cucumeris on snap beans in New York. Phytopathology 85 (12):1554. Agrios, G. N. 2005. Plant pathology Fifth edition ed. New York: Elsevier Academic Press. Akaike, H. 1974. New look at statistical-model identification. IEEE Transactions on Automatic Control 19 (6):716-723. Andersen, T. F. 1996. A comparative taxonomic study of Rhizoctonia sensu lato employing morphological, ultrastructural and molecular methods. Mycological Research 100:1117-1128. Anderson, N. A. 1982. The genetics and pathology of Rhizoctonia solani. Annual Review of Phytopathology 20:329-347. Baker, K. F. 1970. Types of Rhizoctonia solani diseases and their occurrence. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press. Brunner, E., S. Domhof, and F. Langer. 2002. Nonparametric analysis of longitudinal data in factorial experiments. New York: John Wiley & Sons. Buddemeyer, J., B. Pfahler, J. Petersen, and B. Marlander. 2004. Genetic variation in susceptibility of maize to Rhizoctonia solani (AG 2-2IIIB) - symptoms and damage under field conditions in Germany. Zeitschrift Fur Pflanzenkrankheiten Und Pflanzenschutz-Journal of Plant Diseases and Protection 111 (6):521-533. Burpee, L. L., P. L. Sanders, H. Jr Cole, and R. T. Sherwood. 1980a. Anastomosis groups among isolates of Ceratobasidium cornigerum and related fungi. Mycologia 72 (4):689-701. Burpee, L. L., P. L. Sanders, H. Jr Cole, and R. T. Sherwood. 1980b. Pathogenicity of Ceratobasidium cornigerum and related fungi representing 5 anastomosis groups. Phytopathology 70 (9):843-846. 57 Carling, D. E. 1996. Grouping of Rhizoctonia solani by hyphal anastomosis reaction. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. Jabaji-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Carling, D. E., S. Kuninaga, and K. A. Brainard. 2002a. Hyphal anastomosis reactions, rDNA-internal transcribed spacer sequences, and virulence levels among subsets of Rhizoctonia solani anastomosis group-2 (AG-2) and AG-BI. Phytopathology 92 (1):43-50. Carling, D. E., S. Kuninaga, and K. A. Brainard. 2002b. Hyphal Anastomosis Reactions, rDNA-Internal Transcribed Spacer Sequences, and Virulence Levels Among Subsets of Rhizoctonia solani Anastomosis Group-2 (AG-2) and AG-BI. Carling, D. E., R. E. Baird, R. D. Gitaitis, K. A. Brainard, and S. Kuninaga. 2002c. Characterization of AG-13, a newly reported anastomosis group of Rhizoctonia solani. Phytopathology 92 (8):893-899. Carling, D. E., C. S. Rothrock, G. C. Macnish, M. W. Sweetingham, K. A. Brainard, and S. W. Winters. 1994. Characterization of anastomosis group-11 (AG-11) of Rhizoctonia solani. Phytopathology 84 (12):1387-1393. Ciampi, M. B., E. E. Kuramae, R. C. Fenille, M. C. Meyer, N. L. Souza, and P. C. Ceresini. 2005. Intraspecific evolution of Rhizoctonia solani AG-1 IA associated with soybean and rice in Brazil based on polymorphisms at the ITS5.8S rDNA operon. European Journal of Plant Pathology 113 (2):183-196. Cubeta, M. A., and R. Vilgalys. 1997. Population biology of the Rhizoctonia solani complex. Phytopathology 87 (4):480-484. Donk, M. A. 1956. Notes on resupinate Hymenomycetes - II. The tulasnelloid fungi. Reinwardtia 3 ((3)):363-379. Erper, I., G. Karaca, and I. Ozkoc. 2005. First report of root rot of bean and soybean caused by Rhizoctonia zeae in Turkey. Plant Disease 89 (2):203. Erper, I., G. H. Karaca, M. Turkkan, and I. Ozkoc. 2006. Characterization and pathogenicity of Rhizoctonia spp. from onion in Amasya, Turkey. Journal of Phytopathology 154 (2):75-79. Galindo, J. J., G. S. Abawi, and H. D. Thurston. 1982. Variability among isolates of Rhizoctonia solani associated with snap bean hypocotyls and soils in New York. Plant Disease 66 (5):390-394. 58 Garcia, V. G., M. A. P. Onco, and V. R. Susan. 2006. Review. Biology and systematics of the form genus Rhizoctonia. Spanish Journal of Agricultural Research 4 (1):55-79. Godoy-Lutz, G., J. R. Steadman, B. Higgins, and K. Powers. 2003. Genetic variation among isolates of the web blight pathogen of common bean based on PCRRFLP of the ITS-rDNA region. Plant Disease 87 (7):766-771. Gonzalez, D., M. A. Cubeta, and R. Vilgalys. 2006. Phylogenetic utility of indels within ribosomal DNA and beta-tubulin sequences from fungi in the Rhizoctonia solani species complex. Molecular Phylogenetics and Evolution 40 (2):459-470. Gonzalez, D., D. E. Carling, S. Kuninaga, R. Vilgalys, and M. A. Cubeta. 2001. Ribosomal DNA systematics of Ceratobasidium and Thanatephorus with Rhizoctonia anamorphs. Mycologia 93 (6):1138-1150. Hasegawa, M., H. Kishino, and T. A. Yano. 1985. Dating of the human ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22 (2):160-174. Huber, Don M., and Donald R. Sumner. 1996. Suppressive soil amendments for the control of Rhizoctonia species. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. JabajiHare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Hyakumachi, M., and T. Ui. 1987. Non-self-anastomosing isolates of Rhizoctonia solani obtained from fields of sugar-beet monoculture. Transactions of the British Mycological Society 89:155-159. Hyakumachi, M., A. Priyatmojo, M. Kubota, and H. Fukui. 2005. New anastomosis groups, AG-T and AG-U, of binucleate Rhizoctonia spp. causing root and stem rot of cut-flower and miniature roses. Phytopathology 95 (7):784-792. Hyakumachi, M., T. Mushika, Y. Ogiso, T. Toda, K. Kageyama, and T. Tsuge. 1998. Characterization of a new cultural type (LP) of Rhizoctonia solani AG2-2 isolated from warm-season turfgrasses, and its genetic differentiation from other cultural types. Plant Pathology (Oxford) 47 (1):1-9. Ithurrart, M. E. F., G. Buttner, and J. Petersen. 2004. Rhizoctonia root rot in sugar beet (Beta vulgaris ssp altissima) - Epidemiological aspects in relation to maize (Zea mays) as a host plant. Zeitschrift Fur Pflanzenkrankheiten Und Pflanzenschutz-Journal of Plant Diseases and Protection 111 (3):302-312. 59 Johanson, Andrea, Helen C. Turner, Gareth J. McKay, and Averil E. Brown. 1998. A PCR-based method to distinguish fungi of the rice sheath-blight complex, Rhizoctonia solani, R. oryzae and R. oryzae-sativae. FEMS Microbiology Letters 162 (2):289-294. Johnk, Janell Stevens, and Roger K. Jones. 2001. Differentiation of three homogeneous groups of Rhizoctonia solani anastomosis group 4 by analysis of fatty acids. Phytopathology 91 (9):821-830. Jukes, T. H., and C. R. Cantor. 1969. Evolution of protein molecules. In Mammalian protein metabolism edited by H. N. Munro. New York: Academic Press. Katan, Jaacov. 1996. Soil solarization for the control of diseases caused by Rhizoctonia spp. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control, edited by B. Sneh, S. Jabaji-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Kataria, H. R., and U. Gisi. 1996. Chemical control of Rhizoctonia species. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Dordrecht / Boston / London: Kluwer Academic Publisher. Kuninaga, S., T. Natsuaki, T. Takeuchi, and R. Yokosawa. 1997. Sequence variation of the rDNA ITS regions within and between anastomosis groups in Rhizoctonia solani. Current Genetics 32 (3):237-243. Kuninaga, Shiro, Donald E. Carling, Toru Takeuchi, and Ryozo Yokosawa. 2000. Comparison of rDNA-ITS sequences between potato and tobacco strains in Rhizoctonia solani AG-3. Journal of General Plant Pathology 66 (1):2-11. Kuramae, E. E., A. L. Buzeto, M. B. Ciampi, and N. L. Souza. 2003. Identification of Rhizoctonia solani AG 1-IB in lettuce, AG 4 HG-I in tomato and melon, and AG 4 HG-III in broccoli and spinach, in Brazil. European Journal of Plant Pathology 109 (4):391-395. Kuramae, E. E., A. L. Buzeto, A. K. Nakatani, and N. L. Souza. 2007. rDNA-based characterization of a new binucleate Rhizoctonia spp. causing root rot on kale in Brazil. European Journal of Plant Pathology 119 (4):469-475. Leach, L. D., and R. H. Garber. 1970. Control of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press. Lehtonen, M. J., P. Ahvenniemi, P. S. Wilson, M. German-Kinnari, and J. P. T. Valkonen. 2008. Biological diversity of Rhizoctonia solani (AG-3) in a 60 northern potato-cultivation environment in Finland. Plant Pathology 57 (1):141-151. Li, Wu, and Yan. 1998. Aetiology of Rhizoctonia in sheath blight of maize in Sichuan. Plant Pathology 47 (1):16-21. Maddison, W. P., and D. R. Maddison. 2005. MacClade 4.08: Analysis of phylogeny and character evolution Sunderland, MA: Sinauer, . Manici, L. M., and P. Bonora. 2007. Molecular genetic variability of Italian binucleate Rhizoctonia spp. isolates from strawberry. European Journal of Plant Pathology 118 (1):31-42. Mazzola, M., R. W. Smiley, A. D. Rovira, and R. J. Cook. 1996. Chracterization of Rhizoctonia isolates, disease occurence and management in cereals. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Dordrecht / Boston / London: Kluwer Academic Publisher. McNeill, J., F. R. Barrie, H. M. Burdet, V. Demoulin, D. L. Hawksworth, K. Marhold, D. H. Nicolson, J. Prado, P. C. Silva, J. E. Skog, J. H. Wiersema, and N. J. Turland. 2006. International Code of Botanical Nomenclature (Vienna Code) adopted by the Seventeenth International Botanical Congress. In International Botanical Congress. Vienna, Austria: International Association for Plant Taxonomy Menzies, J. D. 1970. Introduction the 1st century of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press. Moore, Royall T. 1987. The Genera of Rhizoctonia-like fungi: Ascorhizoctonia, Ceratorhiza gen. nov., Epulorhiza gen. nov., Moniliopsis, and Rhizoctonia. Mycotaxon 29:91-99. Moore, Royall T. 1996. The dolipore/parenthesome septum in modern taxonomy. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Naito, S. 1996. Basidiospore dispersal and survival. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. 61 Naito, Shigeo, and Seiji Kanematsu. 1994. Characterization and pathogenicity of a new anastomosis subgroup AG-2-3 of Rhizoctonia solani Kuhn isolated from leaves of soybean. Annals of the Phytopathological Society of Japan 60 (6):681-690. Ogoshi, A. 1987. Ecology and pathogenicity of anastomosis and intraspecific groups of Rhizoctonia solani Kuhn. In Annual Review of Phytopathology, Vol. 25, edited by R. J. Cook. Palo Alto, California: Annual Reviews Inc. Ogoshi, A. 1996. Introduction - The genus Rhizoctonia. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Ogoshi, A., M. Oniki, T. Araki, and T. Ui. 1983a. Anastomosis groups of binucleate Rhizoctonia in Japan and North America and their perfect states. Nippon Kingakukai Kaiho 24 (1):79-88. Ogoshi, A., Oniki M., Araki T., and Ui T. 1983b. Studies on the anastomosis groups of binucleate Rhizoctonia and their perfect states. Journal of the Faculty of Agriculture Hokkaido University 61 (2):244-260. Olaya, G., and G. S. Abawi. 1991. Occurrence of Thanatephorus cucumeris on table beets in New York state. Phytopathology 81 (10):1186. Olaya, G., and G. S. Abawi. 1994a. Characteristics of Rhizoctonia solani and binucleate Rhizoctonia species causing foliar blight and root rot on table beets in New York state. Plant Disease 78 (8):800-804. Olaya, G., and G. S. Abawi. 1994b. Influence of inoculum type and moisture on development of Rhizoctonia solani on foliage of table beets. Plant Disease 78 (8):805-810. Olaya, G., G. S. Abawi, and J. Barnard. 1994. Response of Rhizoctonia solani and binucleate Rhizoctonia to 5 fungicides and control of pocket rot of table beets with foliar sprays. Plant Disease 78 (11):1033-1037. Oniki, M., A. Ogoshi, T. Araki, R. Sakai, and S. Tanaka. 1985. The perfect state of Rhizoctonia oryzae and Rhizoctonia zeae, and the anastomosis groups of Waitea circinata. Transactions of the Mycological Society of Japan 26 (2):189198. Parmeter, J. R., H. S. Whitney, and W. D. Platt. 1967. Affinities of some Rhizoctonia species that resemble mycelium of Thanatephorus cucumeris. Phytopathology 57 (2):218-223. 62 Pope, E. J., and D. A. Carter. 2001. Phylogenetic placement and host specificity of mycorrhizal isolates belonging to AG-6 and AG-12 in the Rhizoctonia solani species complex. Mycologia 93 (4):712-719. Posada, D., and K. A. Crandall. 1998. Modeltest: Testing the model of DNA substitution. Bioinformatics 14 (9):817-818. Priyatmojo, Achmadi, Verma E. Escopalao, Naomi G. Tangonan, Cecilia B. Pascual, Haruhisa Suga, Koji Kageyama, and Mitsuro Hyakumachi. 2001. Characterization of a new subgroup of Rhizoctonia solani anastomosis group 1 (AG-1-ID), causal agent of a necrotic leaf spot on coffee. Phytopathology 91 (11):1054-1061. Reiners, S., and C. H. Petzoldt, eds. 2006. 2006 Integrated crop and pest management guidelines for commercial vegetable production. Ithaca: Cornell University Rinehart, T. A., W. E. Copes, T. Toda, and M. A. Cubeta. 2007. Genetic characterization of binucleate Rhizoctonia species causing web blight on Azalea in Mississippi and Alabama. Plant Disease 91 (5):616-623. Roberts, Peter. 1999. Rhizoctonia-forming fungi. Kew: Royal Botanic Gardens. Ronquist, F., and J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19 (12):1572-1574. Saitou, N., and M. Nei. 1987. The neighbor-joining method a new method for reconstructing phylogenetic trees. Molecular Biology & Evolution 4 (4):406425. Salazar, Oscar, Maria C. Julian, Mitsuro Hyakumachi, and Victor Rubio. 2000. Phylogenetic grouping of cultural types of Rhizoctonia solani AG 2-2 based on ribosomal ITS sequences. Mycologia 92 (3):505-509. Salazar, Oscar, Johannes H. M. Schneider, Maria C. Julian, Jaap Keijer, and Victor Rubio. 1999. Phylogenetic subgrouping of Rhizoctonia solani AG 2 isolates based on ribosomal ITS sequences. Mycologia 91 (3):459-467. Shah, D. A., and L. V. Madden. 2004. Nonparametric analysis of ordinal data in designed factorial experiments. Phytopathology 94 (1):33-43. Sharon, Michal, Shiro Kuninaga, Mitsuro Hyakumachi, and Baruch Sneh. 2006. The advancing identification and classification of Rhizoctonia spp. using molecular and biotechnological methods compared with the classical anastomosis grouping. Mycoscience 47 (6):299-316. 63 Sharon, Michal, Stanley Freeman, Shiro Kuninaga, and Baruch Sneh. 2007. Genetic diversity, anastomosis groups and virulence of Rhizoctonia spp. from strawberry. European Journal of Plant Pathology 117 (3):247-265. Sherwood, R. T. 1970. Physiology of Rhizoctonia solani. In Rhizoctonia solani: Biology and Pathology, edited by J. R. J. Parmeter. Berkeley: University of California Press Sneh, Baruch. 1998. Use of non-pathogenic or hypovirulent fungal strains to protect plants against closely related fungal pathogens. Biotechnology Advances 16 (1):1-32. Sneh, Baruch, L. Burpee, and A. Ogoshi. 1991. Identification of Rhizoctonia species. St. Paul: APS Press. Sneh, Baruch, Suha Jajabi-Hare, Stephen Neate, and Gerda Dijst, eds. 1996. Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control. Boston: Kluwer Academic Publishers. Stalpers, J. A., and T. F. Andersen. 1996. A synopsis of the taxonomy of teleomorphs connected with Rhizoctonia s.l. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. JajabiHare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Stalpers, J. A., T. F. Andersen, and W. Gams. 1998. Two proposals to conserve the names Rhizoctonia and R. solani (Hyphomycetes). Taxon 47 (3):725-726. Sumner, D. R. 1996. Sclerotia formation by Rhizoctonia species and their survival. In Rhizoctonia species: Taxonomy, molecular biology, ecology, pathology and disease control., edited by B. Sneh, S. Jajabi-Hare, S. Neate and G. Dijst. Boston: Kluwer Academic Publisher. Sumner, D. R., and D. K. Bell. 1982a. Crop rotation and yield loss in corn in soil infested with Rhizoctonia solani AG-2 and AG-4. Phytopathology 72 (3):361362. Sumner, D. R., and D. K. Bell. 1982b. Root diseases induced in corn by Rhizoctonia solani and Rhizoctonia zeae. Phytopathology 72 (1):86-91. Sweetingham, M. W. 1989. Fungi associated with root and hypocotyl diseases of seedling lupines in Western Australia Australian Journal of Agricultural Research 40 (4):781-790. 64 Swofford, D. L. 2004. Phylogenetic analysis using parsimony (* and other methods), Version 4.0b10. Sunderland, Massachusetts: Sinauer Associates. Tamura, K. , J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution 24:1596-1599. Thompson, Julie D., Toby J. Gibson, Frederic Plewniak, Francois Jeanmougin, and Desmond G. Higgins. 1997. The CLUSTAL-X windows interface: Flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25 (24):4876-4882. Toda, T., J. M. Mghalu, A. Priyatmojo, and M. Hyakumachi. 2004. Comparison of sequences for the internal transcribed spacer region in Rhizoctonia solani AG 1-ID and other subgroups of AG 1. Journal of General Plant Pathology 70:270-272. Toda, Takeshi, Toshihiro Hayakawa, Joseph Mghalu, Shigeharu Yaguchi, and Mitsuro Hyakumachi. 2007. A new Rhizoctonia sp. closely related to Waitea circinata causes a new disease of creeping bentgrass. Journal of General Plant Pathology 73 (6):379-387. Tomaso-Peterson, M., and L. E. Trevathan. 2004a. Rhizoctonia solani AG-13 isolated from corn in Mississippi. Plant Disease 88 (8):908-908. Tomaso-Peterson, M., and L. E. Trevathan. 2004b. Rhizoctonia solani AG-13 Isolated from Corn in Mississippi. Tomaso-Peterson, M., and L. E. Trevathan. 2007. Characterization of Rhizoctonia-like fungi isolated from agronomic crops and turfgrasses in Mississippi. Plant Disease 91 (3):260-265. Tu, C. C., and J. W. Kimbrough. 1975. Morphology, development, and cytochemistry of hyphae and sclerotia of species in Rhizoctonia complex. Canadian Journal of Botany-Revue Canadienne De Botanique 53 (20):2282-2296. Tu, C. C., and J. W. Kimbrough. 1978. Systematics and phylogeny of fungi in the Rhizoctonia complex. Botanical Gazette 139 (4):454-466. Tu, C. C., J. W. Kimbrough, and H. C. Aldrich. 1977. Cytology and ultrastructure of Thanatephorus cucumeris and related taxa of Rhizoctonia complex. Canadian Journal of Botany-Revue Canadienne De Botanique 55 (18):2419-2436. Tu, Chin-Chyu, Ting-Fang Hsieh, and Yih-Chang Chang. 1996. Vegetable diseases incited by Rhizoctonia spp. In Rhizoctonia species: Taxonomy, molecular 65 biology, ecology, pathology and disease control., edited by B. Sneh, S. JabajiHare, S. Neate and G. Dijst. Boston Kluwer Academic Publisher. Vilgalys, R., and M. A. Cubeta. 1994. Molecular systematics and population biology of Rhizoctonia. Annual Review of Phytopathology 32:135-155. White, T. J., T. D. Bruns, and L. D. Leach. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR protocols: A guide to methods and applications., edited by M. A. Innis, D. H. Gefland, J. J. Sninsky and T. J. White. San Diego: Academic Press. Win, H. H., and D. R. Sumner. 1988. Root rot induced in snap bean by Rhizoctonia solani AG-4 and AG-2 type 2 in conservation tillage following corn. Plant Disease 72 (12):1049-1053. Yang, C. S., and R. P. Korf. 1985. Ascorhizoctonia new-genus and Complexipes emend., two genera for anamorphs of species assigned to Tricharina discomycetes. Mycotaxon 23:457-482. Yokoyama, K., and A. Ogoshi. 1988. Studies on hyphal anastomosis of Rhizoctonia solani. V. Nutritional conditions for anastomosis. Nippon Kingakukai Kaiho 29 (2):125-132. 0 66