Session 1: Taxonomy and Identification
Anastomosis groups and subsets of anastomosis groups of Rhizoctonia solani
Molecular phylogeny of Ceratobasidium spp. (BNR) based on rDNA-ITS sequences
Seamless cellulose tubing for the observation of anastomosis of Rhizoctonia solani.
Anastomosis grouping of Rhizoctonia solani isolates from turfgrass based on ITS sequencing
Exploration of pathogenicity and DNA variation of Rhizoctonia solani AG-4
Pathogenicity and anastomosis groups of Rhizoctonia isolated from potato tubers in Argentina
Rhizoctonia species pathogenic of rice in Argentina
Distribution of anastomosis groups of Rhizoctonia solani on potato in Mexico
Characterization of Rhizoctonia solani occurring in sugar beet in the Netherlands
The anastomosis groups of Rhizoctonia solani Kuhn from Taiwan
Occurence of anastomosis groups of Rhizoctonia spp. isolated from different plants in Turkey
Session 2: Population Biology and Genetics
Population biology and genetics of Rhizoctonia solani anastomosis group 3
Structural and functional analysis of hairpin loop DNA plasmid from Rhizoctonia solani
RAPD-PCR used to support concept of sub-populations within Rhizoctonia solani AG-8
Seedling blight of calla lily caused by Rhizoctonia solani in Taiwan
The diversity of the rice sheath blight pathogens in Taiwan
Phylogenetic relationships of Rhizoctonia solani AG4 based on ITS ribosomal DNA sequences.
Function of mitochondirial hairpin loop DNA plasmid pRS224 from Rhizoctonia solani AG2-2-IV
Linear DNA plasmids in Taiwan Rhizoctonia solani AG4 isolates
Widespread distribution of plasmid-like DNAs of Rhizoctonia solani AG-4 isolates in Taiwan
Cloning and characterization of the Rhizoctonia solani AG4 exo-cellulase gene
Session 3: Symbiosis and Plant Fungus Interactions
Symbiotic abilities of mycorrhizae isolated from terrestrially grown and epiphytic orchids
Effects of mycorrhizal infection on different stages of tissue-cultured Dendrobium sonia
Is melanin biosynthesis essential for pathogenicity of Rhizoctonia spp.?
Rhizoctonia-like fungi associated terrestrial orchids in Thailand
Session 4: Ecology and Epidemiology
Epidemiology of rice sheath blight caused by Rhizoctonia solani Kühn under tropical conditions
Three seedling diseases of bitter gourd-loofah graft and their control
Web blight of Urdbean and Mung bean in North India
The occurrence situation of rice sheath blight in Taiwan and pathogen survival tests
Anastomosis groups of Rhizoctonia solani isolates from potatoes in Pakistan
Population dynamics and survival of Rhizoctonia solani AG-1 in field soil
Non target effect of agro chemicals on Rhizoctonia sp.
Rhizoctonia in cereals in Venezuela
Seedling blight of some cucurbitacious crops caused by Rhizoctonia solani
Development of the International Rhizoctonia Committee website
Session 5: Diagnosis and Detection
Trials and tribulations of DNA-based detection of Rhizoctonia solani AG-8 in soil
A specific and quantitative assay for the detection of Rhizoctonia solani AG-3 from potato and soil
Detection of the pathogen causing Eradu patch of lupins
Session 6: Breeding and Induced Resistance
Sensitivity of Rhizoctonia solani to derivatives of natural resorcinols of plant origin
Molecular approaches for the management of rice sheath blight
Induction of chitinase among rice cultivars infected with Rhizoctonia solani
Relationship between resorcinol content in seeds of barley and seedling infection by Rhizoctonia
Preliminary study on resistance to sharp eyespot (Rizoctonia cerealis) in wheat germplasm
Evaluation of inoculation methodologies of Rhizoctonia aerial blight of soybean in Brazil
Cloning chitinase genes for biological control of Rhizoctonia solani and induced resistance
Etiology and chemical control of Rhizoctonia seedling blight and root rot in chickpea
Towards an integrated control of Rhizoctonia solani in sugar beet
Biological control of root rot of wheat caused by Rhizoctonia sola ni
Biological control of Rhizoctonia-disease (Rhizoctonia solani Kühn) of cropped plants in Romania
Management of banded leaf and sheath blight of maize using Pseudomonas fluorescens
Efficacy of some fungicides in controlling root rot of French bean caused by Rhizoctonia solani Kuhn
Situation and Control of Vegetable Diseases Caused by Rhizoctonia spp. in Hubei Province
Occurrence and control of rice sheath blight in Korea
Sensitivity of Rhizoctonia solani towards sheep urine
Rhizoctonia-suppressive composts and potting soil
Studies on bioactivities and antibiotics produced by Paecilomyces marquandii CF110
University of Alaska Fairbanks, 533 East Fireweed Avenue, Palmer, Alaska, USA
Presently there are 14 anastomosis groups (AG) of Rhizoctonia solani described in the literature; AG-1 through AG-13 plus AG-BI. Also, subsets of most of these 14 AG have been characterized using criteria ranging from colony morphology and host range to pectic zymograms to DNA sequences. At least eight subsets of AG-2 have been characterized using combinations of criteria and five subsets of AG-8 have been identified using zymogram patterns.
When different methods are used to characterize a collection of isolates representing a single AG, resulting subsets often are not the same. For example, subsets identified on the basis of host range or virulence may not necessarily match subsets identified on the basis of DNA sequence.
Anastomosis reaction alone often does not provide enough information to allow correct placement of an isolate into an AG. Members of AG-BI (the bridging isolate AG) can easily be mistaken for members of other AG, and certain isolates of many different AG (ie, AG-2, -3, -8, and -11) will react with certain isolates of many other AG. Grouping rDNA sequence seems to be a very reliable way to confirm the AG affinity of questioned isolates.
1Centro Nacional de Biotecnología (CSIC-UAM). Campus Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain; 2Faculty of Agriculture. Gifu University, 1-1 Yanagido, Gifu 501-11, Japan; 3Dept. of Plant Sciences and Institute for Nature Conservation Research, Tel Aviv University, Ramat Aviv, Israel 69978.
The form-genus Rhizoctonia is considered to be an heterogeneous assemblage of fungal taxa, which do not form asexual spores, but have certain significant vegetative characteristics in common. At least 120 epithets referring to Rhizoctonia have been reported, while only few have attempted to clearly suggest an establishment of clear and profound concepts for genera and species for the complex diversity of fungi that are currently considered to belong to Rhizoctonia s.l. A major taxonomic feature is the presence of: multinucleate, binucleate or uninucleate cells in young vegetative hyphae. Binucleate and uninucleate Rhizoctonia species were reported to belong mainly to the teleomorph genus Ceratobasidium Rogers. Another group of binucleate Rhizoctonia, Epulorhiza Moore (=Rhizoctonia repens Bernard) belongs to the teleomorph genus Tulasnella Schroeter.
A basic major obstacle in studying the genetics and taxonomy of Rhizoctonia s.l. is the difficulty, or frequent inability to obtain progeny of the sexual mating and reproduction of many isolates. Therefore, most studies were carried out with cultures of the anamorphs, where the sexual reproductive structures were usually not, or hardly observed, and determination of species was thus based on the morphological and physiological features of the anamorphs. The genera Ceratobasidium Rogers and Thanatephorus Donk were considered by some investigators to be genetically closely related and form a generic complex. The anamorphs of Rhizoctonia-like fungi from these two genera have been traditionally classified on the basis of the number of nuclei per young hyphal cells, considering Thanatephorus as multinucleate and Ceratobasidium as binucleate, although the nuclear position or teleomorph state for many taxa within these two genera still remains unknown.
Certain fungal taxonomists, suggested to integrate several approaches such as: morphometrical, cultural, biochemical, ecological and molecular data, in order to overcome the obstacles, which interfere with taxonomical determinations in Rhizoctonia. This kind of approach may pave the way for establishing a more accurate classification within this complex group of fungi. Consequently, molecular phylogeny of binucleate Rhizoctonia isolates, based on sequences data of ribosomal ITS regions was studied. Fifty-five isolates were analyzed, including tester representatives of most of the currently established binucleate Rhizoctonia anastomosis groups and additional sequences of Ceratobasidium isolates. Two ITS sequences of Waitea circinata and one of Sebacina vermifera, from the GenBank were also included in this study. Agaricus bisporus was used as an outgroup representative. Staining methods were used to determine cell nuclear numbers. Hymenia formation was investigated using new, as well as previously described methods. Data summarized in the phylogenetic trees and clustering analyses indicated that the highly protective non-pathogenic Rhizoctonia (np-R) isolates 521 and Rh2815, which were previously described as multinucleate R. solani AG 4, were clustered together with all the BNR isolates. Nuclear staining confirmed the binucleate status of these isolates. Two binucleate protective isolates that were considered to be BNR, were predicted by comparison of their sequence with available data bases to be closely related to Agaricales (Fam. Tricholomataceae) and morphological examination confirmed this finding. The phylogenetic trees and other results of this study will be discussed in the presentation.
Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, R. O. C.
A high frequency of anastomosis of Rhizoctonia spp. was observed when two isolates of the same AG group were paired grown on seamless cellulose tubing (SCT) with underneath 2% water agar in a moisture chamber for 24 hr at 25 ±2 C. Approximately 30% of hyphae of R. solani contacted had anastomosis that was far higher than those employed on traditional clean slide or water agar technique. The frequency of anastomosis was further improved when 2% of water agar was substituted by 2% dextrose agar. The suitable pH ranges of water agar were between pH 5-8. The reason for higher frequency of anastomosis observed on SCT seems to be that most hyphae were grown on the same plane that make more opportunities for them to attract and contact each other. To prove this hypothesis, different thickness of water agar was prepared, and the results were concord that thicker the water agar less frequency of anastomosis. SCT or thin water agar (less than 100 µm) was the excellent material that could be used for anastomosis observations. By using these materials, there were almost no aerial mycelia to interfere microscopic focusing and observations.
University of Guelph, Dept. Env. Biol., Guelph, Ontario, Canada, N1G 2W1
The internal transcribed spacer region of the ribosomal DNA from four isolates of Rhizoctonia solani from Poa annua and one from Agrostis palustris were sequenced, and compared to sequences from other R. solani isolates representative of 12 anastomosis groups (AG), with multiple sequences from AG1, AG2-1, AG2-2 and AG4. The sequence alignment of the 789 bp region was subjected to distance and parsimony analyses. Both analyses showed that representatives of the different anastomosis groups clustered separately, although branching patterns were not exactly the same in the two dendrograms. Isolates from P. annua all clustered with AG4 HGII. An isolate from A. palustris in Ontario clustered with AG2-1, while an isolate from the same host in Wisconsin clustered with AG1-IB. Screening of a larger number of turfgrass isolates from a wider geographic collection may reveal whether P. annua is always associated with AG4 HGII, and which group or subgroup A. palustris is most frequently associated with.
Department of Botany, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C.
Rhizoctonia solani Kuhn is a worldwide soil-borne pathogenic fungus, which shows tremendous variation in characteristics such as geographic location, morphology, host specificity and pathogenicity. The pathogenicity and DNA variation relationship of fifteen Rhizoctonia solani isolates belonging to the anastomosis group (AG) 4 from Taiwan and one isolate belonging to HGI were examined. The virulence of each strain was tested in loose-leaf lettuce seedlings. The results indicated that of the fifteen strains, eight strains were designated hypervirulent, two strains were middle virulent, four strains were hypovirulent and two strains were non-virulent. The genetic variation in these strains was evaluated using random amplified polymorphic DNA (RAPD) technology with a set of twelve primers. The amplification results revealed scorable polymorphisms among the isolates. Four hundred sixty-seven band positions was scored with binary coding and assayed for Numerical Taxonomy and Multivariate Analysis System (NTSYS) software. The analysis results indicated that the DNA banding patterns of the highly virulent isolates were highly similar to one another. Approximately 650 to 750 base pairs of ITS1-5.8S rDNA ITS2 fragments were amplified using universal primers ITS1 and ITS4 of the fungal ribosomal DNA. The fragments were subjected to restriction digestion with recognition endonucleases EcoRI, HaeIII, MboI and different riboprinting patterns were displayed. The virulence of the sixteen isolates can be related to the differences in the size of the ITS1-5.8S rDNA ITS2 fragments and riboprinting patterns.
1Faculdade de Ciencias Agronomicas/UNESP, CP 237, 18603-970, Botucatu, SP, Brazil; 2Fellowship (FAPESP 97/01098-0)
Rhizoctonia solani is frequently associated with damping-off and foliar blight of the soybean in Brazil. The purpose of this work was to evidence the association of AGs of R. solani with soybean in Brazil, because this pathogen is the most important to soybean crop, essentially in North and Northeast Brazil. Among 73 Rhizoctonia isolates examined, according to nuclear condition, 6 were binucleate and 67 were multinucleate. The multinucleate isolates (R. solani) were characterized according to anastomose group. Among five isolates, causing of the damping-off, one isolate belonged to AG 4 HGI, three to AG 4 HGII and one to AG2.2 IIIB. The others 62, causing of the foliar blight, belonged to AG 1 IA. Molecular marker (RAPD) with four different primers analyzed all R. solani isolates of the soybean and anastomosis groups testers. A total of 35 polymorphic bands were scored for RAPD analysis and by UPGMA clustering three main groups were observed. The first containing all isolates of the foliar blight and AG 1 IA testers. The second containing one among five isolates of the damping-off and AG 2.2 IIIB testers. The third containing the others isolates of the damping-off and both AG 4 HGI and AG 4 HGII. The pathogenicity of those R. solani isolates from soybean was determined under greenhouse condition. The AG 4 HGI, AG 4 HGII and AG 2.2 IIIB R. solani isolates from soybean, were pathogenic in seedlings, causing damping-off and hypocotyl rot. All isolates AG 1 IA from soybean were pathogenic in adult plant, causing foliar blight.
Dept. of Biology, University of Isfahan, Isfahan, IRAN
It is believed that pectic enzyme secretion in fungi could be related with pathogenecity and physiological disorders. It seems that the combination of isoenzyme and its amount of secretion could influence disease severity. The pectic zymograms have been also used to determine the genetic variation and to identify species and pathogenic strains of R. solani. Rhizoctonia disease is one of the most important fungal diseases in potato and bean fields in IRAN. AG-3 and AG-4 isolates of R. solani were isolated from diseased potato and bean plants respectively. Pectic enzyme electrophoresis was performed for 347 AG-3 and 163 AG-4 isolates. The obtained electrophoretic patterns for AG-3 and AG-4 isolates were grouped into three and six zymogram groups (ZG) respectively. The pathogenecity tests were also conducted at glasshouse conditions in a complete randomised block design. The representative isolates belonging to the different ZGs were used as treatments. The data were analysed using Friedman test. The results showed that each ZG causes specific symptoms with varied severity. Isolates belonging to some ZGs were highly pathogenic for AG-3 and AG-4 whereas some others produced very weak or no symptoms. The results indicated that there is genetic variation within AG-3 and AG-4 and also there is varied severity of disease for different isolates of these AGs. However, variation of pathogenecity could not be detected based on anastomosis determination, but ZGs showed a relation between pectic enzyme activity and pathogenecity. Therefore it is concluded that ZG markers could be used to study the severity of rhizoctonia diseases on different hosts.
1Instituto de Ecología, AC., Sistemática Vegetal, Apdo. Postal 63, Xalapa, Ver. 91000, México; 2North Carolina State University, Department of Plant Pathology, Plymouth, NC 27962; 3Duke University, Department of Botany, Durham, NC 2770-0338.
Phenotypic variation among isolates belonging to the Rhizoctonia solani species complex has led plant pathologists to recognize 13 or more anastomosis groups (AG) based predominantly on hyphal anastomosis criteria. However, questions remain concerning whether each AG is a taxonomic unit, and whether these units are species or divergent populations from a single species. In this study a 900 base pair region of the LSU for 46 selected isolates was also sequenced along with the ITS regions of rDNA for 71 isolates representing recognized AG of R. solani and binucleate Rhizoctonia. This data set was complemented with an even larger number of additional ITS sequences from GeneBank. Parsimony analyses showed little if any support for traditional groupings of binucleate and multinucleate isolates, except AG1, AG4 and AG6 of R. solani. Two clades consisting of AG2-1 + AG9, and AG8 + AGBI had a high level of support in most analyses. The remaining AG of R. solani (e.g., AG3, AG5, AG7, AG10 and AG11) did not form stable clades. These results suggest that only AG1, 4 and 6 of R. solani represent well supported taxonomic groups that can be recognized as species.
1Health Sciences University of Hokkaido, Tohbetsu, Hokkaido 061-0293, Japan; 2University of Alaska Fairbanks, 533 E. Fireweed, Palmer 99645, AK, USA
Similarities and differences among 37 isolates of Rhizoctonia solani AG-2 (subgroups 2-1, 2-2, 2-3) and AG-BI , collected from disparate geographic origins, were studied using rDNA sequencing, anastomosis reactions, and pathogenic potential. Relationships were inferred from the sequences of ITS-rDNA. The sequence similarity in the ITS regions was high (93 to 100 %) among isolates within each of the subgroups of AG-2 and AG-BI, but was low (71 to 93 %) between isolates from the different subgroups of AG-2, as well as between isolates from AG-2 and AG-BI. The phylogenetic analysis based on the ITS regions revealed four significantly different clusters corresponding with the three AG-2 subgroups and AG-BI. The analysis of ITS regions confirmed the division of the previously established types within AG-2-2 into the three ecological types (IIIB, IV, and LP). The genetic analysis indicated that AG-2-1 was further divided into at least three types (provisional types: I, II and III) which may differ in cultural morphology and optimal temperature for growth. Type I includes Japanese AG-2-1 isolates and the Dutch AG-2-t isolates (R002, R105 and R144). Type II includes the Alaskan isolate (ATCC 62805) and the Australian isolates (88-033 (ZG-5) and ATCC 44658), and type III includes the Nt-isolate (RT-23) from Italy. We have designed specific primers for PCR identification at the level of anastomosis group (AG-BI), the subgroups (AG-2-1, AG-2-2, AG-2-3) and the ecological types (IIIB, IV and LP) within AG-2-2. Grouping based on rDNA sequence, anastomosis reaction and pathogenic potential will be discussed.
1IMYZA, INTA, CC 25 (1712) Castelar, (Bs.As.) Argentina; 2Facultad de Agronomia, Universidad de Buenos Aires, Argentina
Potato crop in Argentina is affected by several pathogenic fungi, specially Rhizoctonia complex. The purpose of this study was to characterize strains of Rhizoctonia isolated from potato tubers from different regions in Argentina and to determine their pathogenicity. Tubers were collected from fields located in different areas in the provinces of Mendoza, Córdoba and Buenos Aires and examined for sclerotia and mycelia occurrence. Cultures were characterized by anastomosis reaction, performed by pairing isolates with representative testers. Molecular characterization through Polymerase Chain Reaction (PCR) and Restriction Fragment Lenght Polymorphism (RFLP) was also carried out. The isolates were tested for pathogenicity on potato sprouting seed pieces. Controls consisted of uninoculated sprouting seed pieces. Pots were placed in an incubator for 6 wk. Then sprouts were rated on 0-4 scale. Isolates from Córdoba fields were identify as binucleate Rhizoctonia. One isolate from fields located in Buenos Aires was AG-2-1 and the remainder belonged to AG-3. Isolates from tubers collected in Mendoza fields were AG-3. Pathogenicity tests showed that isolates AG-3 were highly pathogenic, midly and nonpathogenic (one isolate) Binucleate isolates and R. solani AG-2-1 were midly pathogenic on sprouts .No lesions were formed on uninoculated control plants. This is the first report of R. solani AG-2-1 on potato in Argentina.
1Cátedra de Fitopatología, Facultad de Ciencias Agrarias, Universidad Nacional del Nordeste, Corrientes, Argentina; 2Instituto de Microbiología y Zoología Agrícola, IMYZA, INTA Castelar, Buenos Aires, Argentina
Spotted leaf sheaths are the most frequent abnormality on rice in the Northeast of Argentina. In this paper we report the results of the research on Rhizoctonia spp., causal agents of the disease. Samples of crops from Corrientes, Chaco and Formosa Provinces were studied using techniques for phytopathogenic fungi. Isolations were identified by morphometric and cultural characteristics and number of nuclei. Pathogenicity was tested by sowing rice seeds on inoculated soil with every Rhizoctonia identified. Three species were found: R. oryzae observed since 1986, is the most frequent in the three Provinces, it causes the “sheath spot”; R. solani detected since 1995 in the same Provinces, is the causal agent of the “sheath blight”; and R. oryzae-sativae found since 1995 in Corrientes, that causes “aggregate sheath spot”. These diseases become evident from the middle of tillering stage and it is difficult to differentiate them in the crop, because of similarity of symptoms. Only sclerotial anamorphs of the three pathogens were found. R. solani and R. oryzae-sativae were detected on leaf sheaths of a rice weed (“red rice”, Oryza sativa f. spontanea), and pathogenicity was proved on it. Increase of the diseases caused by Rhizoctonia spp., observed during the last crop seasons, are due to the introduction of new susceptible semidwarf cultivars, the change of cultural practices and favorable environmental regional conditions.
1Faculdade de Ciencias Agronomicas/UNESP, CP 237, 18603-970, Botucatu, SP, Brazil; 2Fellowship (FAPESP 95/9629-9)
Rhizoctonia solani Kühn has been observed causing damping-off in coffee seedling nursery in different regions in Brazil. Large losses of seedlings occur mainly in the early seedling stage development. The aim of the study was to determine the anastomosis group, genetic variability and pathogenicity of two isolates C1 and C2 of coffee seedlings. The characterization studies were done by cytological character and molecular marker. The number of nucleus per cell was determined through nucleus safranine staining. The anastomosis group characterization was done by hyphae anastomosis with the 12 different AG testers. Once the AG was determined all subgroups belonging to the AG was tested to determine the AG subgroup. Genetic variability between the two isolates was observed by RAPD with 5 primers. The pathogenicity test was carried out in seedlings using soil substrate with R. solani isolates. All isolates of coffee were multinucleate belonging to R. solani. The isolate C1 was anastomosed to AG4 HGII and the C2 to AG4 HGI and AG4 HGII. Thirty-nine polymorphic bands were generated using RAPD marker. A similarity of 85% was observed between both isolates, and 73% similarity between the two isolates and AG4 HGII tester. All isolates were pathogenic to coffee seedlings causing lesions on the hypocotil region.
1Departmento de Produccion Agricola CUCBA Universidad de Guadalajara Apdo, Postal 129, CP. 45110, Guadalajara Jal, Mexico; 2Departamento de Biotecnologia y Bioquimica CINVESTAV Irapuato, A.P. 629, CP. 36500, Irapuato, Gto. Mexico; 3University of Alaska Fairbanks, Agricultural and Forestry Experimental Station, 533 East Fireweed, Palmer 99645, USA.
The main potato growing regions in Mexico are Guanajuato, Sinaloa, Sonora, Estado de Mexico, Puebla, Coahuila, Chihuahua, Jalisco, Michoacan and Veracruz. Approximately 65,000 hectares of potatoes are planted in Mexico each year and Rhizoctonia solani is an important disease in the most of Mexico’s potato growing regions. In this work we report the anastomosis group (AG) identity of R. solani isolates collected from potatoes growing at various locations in Mexico. Of 68 isolates collected near Leon in Guanajuato, 50 were AG-3 and 18 were AG-4, Sixteen of 20 isolates collected in Sinaloa were AG-3 and 4 were AG-4. Twenty four isolates collected in Sonora all were members of AG-3. Similarly, 40 isolates collected in Jalisco and 20 collected in Veracruz all were AG-3. In Estado de Mexico 51 of 57 isolates were AG-3 and the remaining six were AG-7. All of the AG-7 isolates were found in the vicinity of Temascaltepec. In the Puebla region we found two isolates of AG-4 and 8 isolates of AG-3. In Coahuila and Nuevo Leon, 90% of all isolates collected were AG-3, 5% were AG-4, and the other 5% were either AG-2 and AG-5.
Institute of Sugar Beet Research (IRS), P.O. Box 32, 4600 AA Bergen op Zoom, the Netherlands
Rhizoctonia solani causes severe crop loss in sugar beet in the Netherlands. Symptoms include black root rot of seedlings, wilting, crown and root rot. Anastomosis groups isolated from infected beets include AG 1-IC, AG 2, AG 3, AG 5, some unidentified R. solani isolates and Waitea spp. The dominant pathogen AG 2-2IIIB, was isolated from sugar beet throughout the growing season. Under standardized conditions AG 2-2IIIB isolates infected sugar beet seedlings at 23 ºC, but inoculum from the same batch did not cause disease when tested simultaneously at 10 ºC. Isolates of AG 2-2IIIB varied in aggressiveness when tested in the same experiment at 23 ºC or when tested on 8-week old plants. R. solani AG 2-2IIIB was also isolated from field grown carrots, black salsify, fodder beet, lily, gladiolus, Angelica sylvestis, Levisticum officinale and volunteer potatoes. Furthermore, AG 2-2IIIB was isolated from roots of maize and rye grass of apparently healthy plants. AG 2 isolates were also characterised by pectic zymography, ITS - RFLP and various RAPD primers. The latter technique may yield specific primers for detection of AG 2-2IIIB in infected beets or bio-assays. AG 3 isolates hampered growth of seedlings in the field, caused lesions on mature beets, and formed the perfect stage, Thanatephorus cucumeris, on the stems of the leaves. AG 3 isolates did not cause yield reduction in commercial fields.
1Department of Plant Pathology, Taiwan Agricultural Research Institute, Wu-feng, Taichung 413, Taiwan, R.O.C; 2Retired from former Department of Agriculture and Food, Taiwan provincial government, Taipei, Taiwan, R. O. C.
Root tissues of 65 species of crops distributing in 29 families were collected. After surface sterilization with 1% sodium hypochloride for 3 min and rinsing in sterile distilled water 3 times, the tissues were cutted and placed on 2% Difco bacto-agar supplemented with 100 µg/ml of streptomycin sulfate for isolation of Rhizoctonia-like fungi(RLF). A total of three hundred and forty-nine isolates of RLF were obtained. Among those isolates, hyphal tip cells of 61 isolates were dinucleate and 288 isolates were multinucleate. Among multinucleate RLF, 262 isolates were identified as Rhizoctonia solani, 13 isolates were Rhizoctonia zeae, 1 isolate was Sclerotium hydrophilium and 12 isolates were unknown. Of total 262 isolates of R. solani, 24 isolates were belong to AG-1, 13 isolates were AG-2, 4 isolates were AG-3, 170 isolates were AG-4, 20 isolates were AG-7 and 31 isolates failed to anastomose with all of the 11 standard AGs testers (AG-1 to AG-10 and AG-BI) provided by Dr. Ogoshi. Four isolates of AG-1, 5 isolates of AG-2, 1 isolate of AG-3, 6 isolates of AG-4 and 4 isolates of AG-7 were selected to test their pathogenicity on 14 species of crops. In the study, the virulence of AG-4 isolates of R. solani was much higher than that of other AGs isolates. AG-4 isolates were able to infect all tested plants and cause severe diseased symptoms. Virulence of Rhizoctonia isolates was different from each other according to the kind of crops were inoculated. Virulence of AG-2 and AG-7 was significantly lower than that of other AGs. In pathogenicity tests, AG-3 isolates obtained from diseased lilies were specific to attack lily plants and cause seedling blight.
1Plant Protection Central Research Institute, Yenimahalle,Ankara,Turkey; 2Department of Plant Patholgy of Ankara University Dışkapı, Ankara, Turkey
This research has shown that 159 Rhizoctonia solani Kühn isolates collected from plants like barley, wheat, potato, green pepper tomato, chickpea, green bean, soja, obacco, sugar beet, alfalfa, carrot, carnation, pear,.strawberry, soil and 2 binucleate Rhizoctonia isolates from pear in Turkey; fell into 8 different anastomosis groups:15 isolates were assigned to be in AG-2 Type-1, 82 in AG-3, 33 in AG-4,15 in AG-5 4 in AG-6, 8 in AG-8 and 2 in AG-BI. 2 binucleate Rhizoctonia isolates fell into AG-K.
All Rhizoctonia(AG)s were examined to determine the protein patterns by using PAGE(Polyacrylamaide gel electrophoresis) tecnique. Besides, Internal transcribed spacer(ITS) and Intergenic spacer(IGS) were studied to amplified.
Centraalbureau voor Schimmelcultures, P.O box 273 3740 AG Baarn, The Netherlands; Plant Research International, P.O. box 9060, Wageningen, The Netherlands.
The species complex Rhizoctonia solani shows a high variation in pathogenicity as shown by the disease severity and host specificity of different strains. The recognition of anastomosis groups (AG) is a generally accepted way of classification below the species level. About twelve different anastomosis groups have been recognized with several subgroups. The AG has a predictive value for host plant groups at risk, which is of economic importance since it can help in agriculture crop selection. However, the AG determination is hampered by several difficulties: high inter-observer-variation, a laborious procedure, availability of specialists and degeneration of tester-strains. In order to get a better understanding of the role of anastomosis and the correlation between genotype, pathogenicity and AG we conducted a pilot study in which we compared AFLP patterns and ITS sequences of 70 different R. solani strains. A secondary goal is to develop a molecular identification system.
1Faculdade de Ciencias Agronomicas/UNESP, CP 237, 18603-970, Botucatu, SP, Brazil; 2Fellowship FAPESP 95/9629-9
Rhizoctonia solani Kuhn is associated to damping-off, leaves necrosis and root, stem and fruit rots. Rhizoctonia solani has been causing leaves necrosis and stems and fruit rots in lettuce and tomato, respectively. The goal of this study was to characterize isolates of lettuce and tomato from different regions in Brazil through nuclei number, hyphae anastomosis and pathogenicity. A total of three isolates of lettuce and five of tomato with leaves necrosis and stem rots, respectively were analyzed. The number of nuclei per cell was determined using safranina staining. The anastomosis reaction was carried out by hyphae anstomosis (Herr & Roberts, Phytopathology, 70:476, 1980) between isolates of lettuce and tomato with 12 different testers. The pathogenity test was done in 40 days old seedlings of lettuce and tomato by inoculating a 0.5 cm disc containing micelia on the leaves of lettuce and stems of tomato. The isolates of lettuce had 8 to 13 nuclei and isolates of tomato had 4 to 7 nuclei per cell those isolates were classified as R. solani. All lettuce isolates were positive reacted to AG1 IA while all tomato isolates reacted to AG4 HGI. The three isolates of lettuce were pathogenic causing leaf necrosis in all plants tested and all isolates of tomato caused stem rot in all tomato plants tested.
1North Carolina State University, Department of Plant Pathology, Raleigh, NC, 27695; 2CNPq/UNESP, Sao Paulo, Brazil; 3Duke University, Department of Botany, Durham, NC 27708; 4USDA-ARS, University of Minnesota, St. Paul, MN 55108; 5North Carolina State University, Department of Plant Pathology, 207 Research Station Road, Plymouth, NC 27962
A polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method (a modification of method previously described by Xu et al., Molecular Ecology 8:59-73) was developed to identify codominant, single-locus genetic markers to examine the diversity and structure in populations of Rhizoctonia solani AG-3 from potato and tobacco in North Carolina. Random pieces of DNA of R. solani AG-3 from potato and tobacco were cloned into pUC18 to transform competent E. coli cells. Clones with an insert size of 1-1.6 kb were selected and sequenced (20 clones each for potato and tobacco). PCR primers (20-30 bp) were developed based on sequence data and used to amplify each selected clones. Each PCR product was digested with multiple restriction enzymes to identify polymorphic restriction sites corresponding to a specific restriction enzyme/primer pair combination. After sequencing and screening anonymous DNA from each genomic library, seven and no polymorphic codominant single-locus PCR-RFLP markers were identified from library containing DNA of R. solani AG-3 from potato and tobacco, respectively. These seven PCR-RFLP markers were used to identify unambiguous genotypes to assess patterns of mating and gene flow (migration) in populations of R. solani AG-3 from commercial potato production fields in five counties in eastern North Carolina. There was evidence for recombination and gene flow within and between populations. Most loci did not depart from Hardy-Weinberg expectations and there was a random association of alleles within and between loci in greater than 92% of the loci or pairs of loci, providing an indication of random mating.
1North Carolina State University, Department of Plant Pathology, 207 Research Station Road, Plymouth, NC 27962; 2North Carolina State University, Department of Plant Pathology, Raleigh, NC, 27695; 3CNPq/UNESP, Sao Paulo, Brazil; 4Duke University, Department of Botany, Durham, NC 27708
The anastomosis group (AG) concept represents the single most important advance in our understanding of the genetic diversity in Rhizoctonia solani, however, little information is available concerning the nature of individuals and populations in most Rhizoctonia pathosystems. Previous studies have not addressed questions concerning patterns of mating (i.e. asexual versus sexual reproduction) and the importance of gene flow (migration) in differentiating populations of R. solani. For the past few years, our laboratory has been examining the genetic diversity and structure of populations of R. solani AG-3 from potato and tobacco using three independent criteria; somatic incompatibility, amplified fragment length polymorphism (AFLP) and PCR-RFLP based analyses. The PCR-RFLP method has subsequently been used to compare the relative contribution of clonality and recombination to the observed population structure of R. solani AG-3 from potato by assessing the association of alleles within and/or between loci in a population genetic framework. Results suggest that populations of R. solani AG-3 from potato (a predominantly asexual pathogen) are more genetically diverse than populations from tobacco (a predominantly sexual pathogen). The tobacco population is clonal in structure, while there was evidence for recombination in the potato population (e.g. many, but few shared multilocus genotypes; no association of alleles at unlinked loci=gametic equilibrium; and no evidence for segregation at individual loci). However, because of the excessive migration of inoculum on potato seed tubers, a clonal component could not be excluded. Experiments are currently in progress to assess the role tuberborne inoculum plays in determine population structure.
Department of Environmental Biotechnology, Graduate School of Agriculture, Tohoku University, Sendai 981-8555, Japan
Hairpin loop linear plasmids were found in the field isolate of Rhizoctonia solani. Considerable sequence homology at the nucleotide level of plasmids and plasmid-encoded transcripts were found among them obtained from different isolates within the same anastomosis group (AG). To know about the function of the plasmids, we analyzed their coding regions and plasmid-encoded proteins. Unique poly(A)-RNA, 0.5 in length which hybridize with the pRS64 DNA, was found in the isolate RI-64 of AG4. The open reading frame (ORF) found in pRS64 is 68 amino acid long and has potential coding capacity of 7 kDa. The amino acid sequence encoded by the ORF showed no significant homology with the known proteins. On the other hand, unique poly(A)-RNAs, 4.7 and 7.4 kb in length which hybridize with the pRS224 DNA, were found in mycelial cells of R. solani H-16 of AG2-2. The 7.4 kb transcript is generated from the left terminal region of the complementary strand. Open reading frame found in pRS224 is 887 amino acids long and has potential coding capacity of 102 kDa. The ORF contains the highly-conserved domains characteristic of reverse transcriptase sequences. The role of the transcript is not yet clear.
Unit of Mycology, Bacteriology and Nematology, Scottish Crop Research Institute, Dundee, DD2 5DA, UK
Black scurf of potato is caused by infection of the tuber surface by Rhizoctonia solani and results in the formation of irregular black sclerotia that affect skin quality. R. solani AG-3 is mainly associated with black scurf, although AG-4, AG-5 and AG-9 have also been observed. In order to breed for resistance to black scurf, it is necessary to examine the level of variation within AG-3. A world–wide collection of 35 AG-3, and 32 other isolates representing 12 groups, was therefore obtained, and assessed for phenotypic and genetic variation.
It was found that cultural morphology, which incorporated assessments of aerial mycelia, substrate colour, area covered in sclerotia and sensitivity to the fungicide pencycuron in amended media, differed between isolates. However, none of these characters were specific to individual anastomosis groups. Pathogenicity tests carried out under controlled environmental conditions revealed that AG-3 isolates varied in pathogenicity, but were more aggressive than isolates of AG-2-1, AG-4 and AG-6 which were also able to cause infection.
Molecular characterisation using cluster analysis based on Amplified Fragment Length Polymorphisms (AFLPs) with different primer combinations, revealed that isolates of AG-3 are distinct from other groups. However, considerable variation exists both within AG-3 and between other groups.
Graduate School of Agriculture, Hokkaido University, Kita-ku, Sapporo, 060-8589, Japan
Little is known about the role of single basidiospore isolates of Thanatephorus cucumeris in determining the genetic diversity of field populations of Rhizoctonia solani in nature. The present study was conducted to examine the somatic interactions of single basidiospore isolates of T. cucumeris (R. solani AG 2-2-IV) within the same and over several generation(s) and to determine their contribution to the population diversity evaluated as colonization or infection of sugarbeet. 1) Laboratory test: in many cases, single basidiospore progeny of each parental isolate from sugarbeet roots was heterogeneous with respect to their somatic interactions in the first generation of teleomorph stage (Gl), while that from infected leaves was homogenous (i.e., vegetatively compatible). Each of heterogeneous sibling basidiospore isolates in the G1 tended to yield homogenous progeny within a few generations. 2) Field test: the parental isolate Pf 28 and the isolate Pf 28S-6-1, consisting of heterogeneous and homogenous progenies in the G1 and G3, respectively, were added to the soil in different sugarbeet fields in early summer, 1997. Foliar blight disease originating from basidiospore infections occurred in the both fields over 2 years. Many pairings of leaf isolates from the infected field with Pf 28 were somatically heterogeneous, while those from the infected field with Pf 28S-6-1 were homogenous. Finally, contribution of basidiospore infections to the observed genetic diversity in field populations of R. solani in sugarbeet will be discussed here.
1Applied and Molecular Ecology, The University of Adelaide, PMB 1, Glen Osmond, South Australia 5064; 2South Australian Research and Development Institute, Waite Precinct, South Australia; 3CSIRO Land and Water, Waite Precinct, South Australia; 4Columbia Basin Agricultural Research Center, Oregon State University, Pendleton, Oregon, USA.
Rhizoctonia solani AG 2 is divided into AG 2-1, AG 2-2 and AG 2-3 based on anastomosis frequency, thiamine requirement and pathogenicity (Carling, 1996). Zymogram analysis has been used to subdivide AG 2-1 into zymogram groups (ZG) 5, including subgroups, and ZG 6, while AG 2-2 is divided into ZG 4 and 10 (MacNish et al., 1994; Schneider et al., 1997). In addition, subgroups of AG 2 can be distinguished on the basis of ribosomal ITS sequences (Salazar et al., 1999). Our aim was to examine genetic diversity among AG 2 isolates and to identify DNA markers specific to this group.
AG 2 isolates from Australia, Japan and the Netherlands were assessed for variation in zymogram patterns using vertical 12% poly-acrylamide gel electrophoresis. The technique separated 25 patterns which were used to group isolates of both AG 2-1 and 2-2 into the known ZG. However, AG 2-1 and AG 2-2 isolates were highly variable and further subdivisions are likely to exist.
Genetic variation among AG 2 isolates was determined using PCR and RFLP analyses. Two microsatellite primers, (CAC)5 and (GACA)4, and the intron-splice junction primer, Rl revealed polymorphisms with reproducible banding patterns and 10 random genomic clones were identified as informative RFLP markers. Both approaches detected high levels of genetic diversity within AG 2 isolates. While no AG 2-specific DNA probes have been isolated, markers which identify AG 2-l but not AG 2-2, and vice versa, but which cross-react with AG 8 and 4 are under investigation.
School of Biological Sciences & Biotechnology, Murdoch University, Perth, 6150, Australia
In Australia a bare patch disease of cereals is caused by Rhizoctonia solani AG-8. It has been established that isolates of AG-8 can be separated into distinct pectic 鵾ymogram’ groups (ZGs) with five ZGs (1-1 to 1-5) being detected. Studies of vegetatively compatible populations (VCPs) within AG-8 have shown that within each ZG there are compatible as well as incompatible populations. VCPs never extended across ZGs. This supports the possibility that ZGs are distinct sub-populations within AG-8.
RAPD-PCR was used to study the genetic diversity of 81 isolates from four ZGs (1-1, 1-2, 1-4 and 1-5) from various locations in Australia. All isolates from each ZG were grouped according to VCPs. RAPD-PCR was performed on all isolates using six primers. Relatedness of the isolates was determined by combining the results using RAPDistance programme version 1.04 to generate a Neighbour Joining tree.
The data showed that the population consisted of four distinct groups that matched the four ZGs. This again supports the concept of ZGs being distinct sub-populations. Within each ZG the isolates were grouped according to genotype. For ZG1-1 and ZG1-4, the genotype groupings showed little or no correlation with the VCPs groupings. But for ZG1-2 and ZG1-5 there was a good correlation between genotype and VCP groupings. The implications of these results will be discussed.
C.W. Chen1, M.G. Chiu2 and L.C. Chen2
1Taichung District Agricultural Improvement Station, Council of Agriculture, Taichung; 2Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, R. O. C.
Calla lily (Zantedeschia) is a new cut flower and potted plant in Taiwan. The seedling blight caused by Rhizoctonia solani can cause serious loss of calla lily plants in the field when it germination. It's another major inhibit factor in the field culture of Taiwan than Erwinia soft rot. The symptom was bud stem and leaf rot or necrosis. Total 26 isolates of Rhizoctonia solani were isolated from infected site of calla lily. Most isolated of R. solani was identified belong to AG-4 anastomosis group with hyphal fusion. The pathogen could be isolated from the base stem or rhizome of calla lily. From the fields collected, pathogens could isolated from the plant of different cultivar such as Z. albomaculata, Black Magic, Fandango, Pink Pear, Florex Gold, Dominque, Magic Red. Observation the resistance ability of 26 cultivars calla lily resistance to the pathogen. The hightly susceptible cultivar to R. solani were Neroli and Treasure. Highly resistance cultivar was Golden Sun and Rose Queen. There was no absolute relationship between spath's color of calla lily and resistance ability to R .solani. The population dynamic of R.solani on calla lily in the field was depended on the temperature of seasons. This was first reported of this disease in Taiwan.
Dept. of Plant Pathology, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, ROC
The sheath blight is still an important one of rice diseases in Taiwan. There were fewer researchers to study this disease during the last 20 years in the island. We are interested in the changes of the disease and want to find the differences between nowadays and the past. So we proceeded the identification of anastomosis group / subgroup, compare the geographical distribution, temperature, or latitude with the colony types, virulence, growth rate, construction of sclerotia, or density of sclerotia, and prove the isolates from different areas using different superiorities to exist in the natural word. Then we use the RAPD technique as a side proof to compare the genetic relationships. We find that the isolates from southern Taiwan are more virulent than those from northern Taiwan. In accordance with predecessors' information AG-1 IB and IC have linear plasmids, but AG-1 IA doesn't. Then the isolates Rs1E-2 and Rs1E-5 should be belong to AG-1 IA according to their sclerotium types, but they both have 2.0kb in total DNA patterns and only fuse with AG-1 IC standard isolate. It is worthy of analyzing continuously. Besides, Rs1E-3 can fuse with AG-1 IA standard isolate and the fuse points are narrower than the diameter of the hypha (C2), but the surface of sclerotia is not smooth and has many aerial hypha. Its sclerotia and colony type are unlike general AG-1 isolates. These phenomenons are very extraordinary and reflect the population complexity of rice sheath blight pathogens in Taiwan.
Dept. of Plant Pathology, 406 Plant Sciences Hall, P.O. Box 830722 Lincoln, Nebraska 68583-0722, USA
Variability of isolates of Thanatephorus cucumeris (FRANK) Donk (Rhizoctonia solani), the web blight pathogen (WB) of dry beans Phaseolus vulgaris L. was examined by PCR-RFLP and sequence of the ITS-5.85 rDNA. Isolates were collected from diseased leaves from Argentina, Costa Rica, Cuba, Dominican Republic, Honduras, Panama and Puerto Rico. These WB isolates belong to AG-1 and AG-2 according to traditional genetics. Isolates of AG-1 that cause WB were separated into three distinct RFLP patterns from enzymatic digestion of 740 bp fragment.
Microsclesotia producing isolates (< 1 mm) were differentiated from macrosclerotia producing isolates (5-20 mm) even though they are placed in the same intraspecific group AG-1-IB. Sequence analysis agreed with the RFLP pattern differences. WB isolates of AG-2were separated into two distinct RFLP patterns. These isolates differ in cutting sites from other AG-2 isolates previously reported.
Department of Botany, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C.
Rhizoctonia solani Kühn, a worldwide soil-borne pathogenic fungus, shows tremendous diversity in its subgroups because of different habitats. Studies on the fungal ribosomal genes (5.8S, 18S, and 28S) have shown that these genes are highly conserved at the genus level, but both ITS regions show variations among strains and can be used to evaluate the genetic relationships. The virulence of fourteen isolates of R.solani, belonging to the anastomosis (group) 4, were grown on PDA and transferred to PDB. The genomic DNA was extracted for the asymmetric PCR template. Approximately 650 to 750 base pairs of ITS1-5.8S-ITS2 fragments were amplified using universal primers ITS1 and ITS4 of highly conserved regions of fungal ribosomal DNA. Sequencing was done with single-stranded PCR products as templates and ITS4 as the sequencing primer.
In results from the alignment of the sequenced R.solani AG-4 strains, thirteen isolates from Taiwan and five isolates from Japan, using the GCG program show that the 5.8S coding region is completely conserved and similar to that in other fungi. Within R.solani AG-4 there is variation in the ITS1 and ITS2 region. A bootstrap analysis using 1000 re-samples of the sequence data was performed. Phylogenetic analyses based on the PAUP and Mega program suggest that the analyzed R.solani AG-4 can be divided into two groups that correlate with habitat and virulence.
1Faculdade de Ciencias Agronomicas/UNESP, CP 237, 18603-970, Botucatu, SP, Brazil; 2Fellowship (FAPESP 97/01098-0)
Rhizoctonia foliar blight (RFB) of soybean is a destructive foliar disease caused by the fungus Rhizoctonia solani Kühn anastomosis group (AG) 1. In Brazil the disease have been important due the expansion of soybean in equatorial area. Yang et al (Plant Dis. 74:501, 1990) showed that RFB on soybean was caused by two types of R. solani AG 1: intraspecific group A (IA) and intraspecific group B (IB). However, field symptoms caused by these types are similar. Our objective was determinate the anastomosis group associated with foliar blight on soybean in Brazil. Sequence analysis of the rDNA region containing the internal transcribed spacer (ITS) regions and the 5.8s rDNA coding sequence was used to compare 19 R. solani isolates, causing of the foliar blight on soybean, and subgroups IA, IB and IC within AG 1 testers. The sequence homology in the ITS regions was above 92% for R. solani isolates from soybean and subgroup IA, 33-38% for R. solani isolates from soybean and subgroup IC, and 35-40% for R. solani isolates from soybean and subgroup IB. The sequence homology in the ITS regions was 34-38% for isolates testers of different subgroups within AG1. The results corroborate to the identification of the R. solani isolates from soybean by hyphae anastomosis methods. AG 1 IA is the anastomosis group associated with foliar blight on soybean in Brazil.
1Faculdade de Ciencias Agronomicas/UNESP, CP 237, 18603-970, Botucatu, SP, Brazil; 2Fellowship (FAPESP 95/9629-9)
The soil-borne plant pathogen Rhizoctonia solani Kühn is a complex specie that is composed by 13 different anastomosis group (AG). The 13 AG groups are morphologically similar. However the AGs differ genetically and thus, in their ability to cause disease in several crops. In Brazil, R. solani causes large losses of bean (Phaseolus vulgaris) and peanut (Arachis hypogaeae) in field production. The aim of this study was to determine the genetic variability among isolates of bean, peanut and AG testers of R. solani and Rhizoctonia spp. binucleate. We have sequenced the internal transcribed spacers (ITS) and 5.8S nuclear rDNA region, and also mitochondrial rDNA of R. solani and Rhizoctonia spp. binucleate isolates associated to bean and peanut and also R. solani and Rhizoctonia spp. binucleate AG testers. The PCR products were obtained by ITS4 and ITS5 primers for nuclear rDNA and, ML3 and ML4 for mitochondrial rDNA. The sequences were aligned and analyzed by OMIGA 2.0 software. Variability in the nuclear and mitochondrial rDNA sequences was observed among isolates of R. solani and among isolates of Rhizoctonia spp. binucleate of the two crops and also among R. solani and Rhizoctonia spp. binucleate AG testers.
Department of Environmental Biotechnology, Graduate School of Agriculture, Tohoku University, Sendai 981-8555, Japan
It is known that hairpin loop DNA plasmids in Rhizoctonia solani has characteristic group sizes within the different anastomosis (AG). To know about the function of the plasmids, we analyzed their coding regions and plasmid-encoded proteins. It has been confirmed that two kinds of transcription sizes measuring 4.7 kb and 7.4 kb exist for the isolate H-16 of AG2-2-IV. As a result of examining the sequence of this plasmid, it has been discovered that protein RS224 consists of 887 amino acids. Anti-pRS224-encoded protein (RS224) antibodies raised against the partial polypeptide of protein RS224 cross-react with the specific protein found in the mycelia. Furthermore, it was revealed by the cell fractionation method that this protein lies mainly in the mitochondrial fraction. This protein, however, has no significant homology with other known proteins. Additionally, this protein contains seven short amino-acids which establish a relationship with reverse transcriptase encoded by some mitochondorial plasmids and group II intron of fungus, as well as some retroviruses.
Department of Botany, National Chung Hsing University, Taichung, Taiwan, ROC
Sixteen plant pathogenic Rhizoctonia solani AG4 isolates were obtained from soils in Taiwan. Eight out of the 16 isolates contained a linear DNA plasmid with a hairpin loop structure at both termini and a molecular size of 2.6 kb. Significant sequence homology was observed among the plasmid DNAs in the representative isolates using Southern blot analysis. The virulent strain (pCHU352) contained an open reading frame (418 bp) that encoded a specific function-unknown protein of 7 kDa. There were no effects on disease severity by plasmid transformation to non-plasmid carrying strains and the protoplasts fusion between plasmid and non-plasmid carrying strains R. solani. These results indicate that no apparent correlation between the mere presence of linear plasmids and the degree of virulence.
Department of Botany, Chung Hsing University, Taichung, Taiwan, ROC.
Pathogenic and non-pathogenic strains of Rhizoctonia solani AG-4 were isolated from central Taiwan. F41, an anastomotic strain, was isolated from CHU341 and CHU344 mating experiments. CHU341 was a pathogenic strain with 2.6 kb plasmid, CI (Conduciveness) index 0.89. CHU344 was a non-pathogenic strain without plasmid, CI index 0.09. F41 was a pathogenic strain with 2.6 kb plasmid and 0.58 CI index. The trasgenic strain of CHU344-ES was a non-pathogenic strain with a 0.09 CI index. The RAPD banding pattern showed that F41 was a genetic recombination strain of CHU344 and CHU341. In the ITS fragment amplification, CHU344 was 620 bp, CHU341 and F41 was 750 bp. The optimum growth rate of the CHU341 strain was at pH 7.8, 25 ℃. Optimum CHU344 growth was at pH7.5, 25.8 ℃. Optimum F41 growth was at pH 7.7, 25. ℃. The physiology and genetic character of F41 was similar to the CHU344 and CHU341 strains with 2.6 kb plasmid. The evidence indicated that the nucleus of the CHU344 strain moved into the CHU341 strain to reduce the pathogenic character of CHU341.
Department of Plant Pathology, Chung-Hsing University, ROC
It is amazing how widely distributed eucarytic plasmid DNA. 72 isolates of Rhizoctonia solani AG-4 were originated from the vegetables and flowers in Taiwan. For investigating the distribution of plasmid-like DNA (plDNA) of R. solani AG-4 isolates in Taiwan, we extracted the total DNA of the 72 isolates and 20 isolates of AG-1~AG-9, and had a analysis on number of plDNA by 1% agarose gel electrophoresis. Among 72 isolates of R. solani AG-4, twenty-four contain one plDNA, twenty-two contain two plDNAs, two contain three plDNAs, and eight contain four plDNAs. The size of these plDNAs were 2.2 to 2.7 Kb. The other sixteen contain no plDNA. The total DNA of the collected R. solani isolates were hybridized with nonradiolabeled plDNAs of AG-4 SH-O-1 originated from cabbage and B sp-5 originated from spinach. We found the plDNAs in the AG-4 isolates have homology with each other, but have no homology with the other anastomosis group. Specially, the plDNAs in the R. solani AG-4 isolates were distinguished into two group according to the result of Southern’s hybridization. We need to further confirm the plDNAs of R. solani are widespread distribution and it’s role on the dynamics of population density of the fungus.
1Supported by CONACyT-Mexico (3230P-B9607); 2Universidad Autonoma de Aguascalientes, Aguascalientes, Mexico, 20100; 3Colegio de Postgraduados, Montecillo, Mexico, 56230; 4Instituto Nacional de Investigaciones Forestales y Agropecuarias (INIFAP) A. Postal 10, Chapingo, Mexico, 56230; 5Centro de Investigacion y de Estudios Avanzados, A. Postal 629, Irapuato, Mexico, 36000
Rhizoctonia bataticola (Macrophomina phaseolina) causes charcoal rot in common bean (Phaseolus vulgaris L.) in Mexico. Analysis of 84 isolates of R. bataticola, using 12 common bean cultivars and AFLP's was carried out. Isolates were obtained from different host species and locations of Mexico. Cluster analysis performed on pathogenicity data and genotype data for a11 isolates showed clear pathogenicity groups although these groups were not found to be associated with host or geographical origins. AFLP genotypes did not show clear groupings. When only the isolates obtained from common bean were analyzed, groups associated with the geographical origin of the isolates were found for both, pathogenicity and genotype data. Groups associated with geographical or host origin of the isolates were also found when the isolates collected within the state of Tamaulipas were analyzed. Specific interactions between common bean cultivars and certain isolates were determined. The disease reaction of 64 common bean cultivars was evaluated under field conditions. Cultivars were established in five experiments in artificially inoculated and control plots at three lowland sites in Mexico. A negative association between disease severity and seed yield was observed in a11 sites. Resistant cultivars were mainly ascribed to the lowlands Mesoamerican race, while susceptible cultivars mainly were from the highlands Durango and Jalisco races. This information will be a useful base for determining breeding strategies and for genetic analysis of resistance to charcoal rot in common bean for Mexico.
1North Carolina State University, Department of Plant Pathology, Raleigh, NC, 27695; 2CNPq/UNESP, Sao Paulo, Brazil; 3Duke University, Department of Botany, Durham, NC 27708; 4North Carolina State University, Department of Plant Pathology, 207 Research Station Road, Plymouth, NC 27962
Rhizoctonia solani AG-3 is important pathogen of several different cultivated Solanaceous hosts including eggplant, potato, tomato and tobacco. Previous studies indicate that isolates of R. solani AG-3 are taxonomically related based on analyses of ribosomal DNA (rDNA) genes, but exhibit considerable differences in ecology, epidemiology, host specificity and reproductive biology. In this study, the genetic diversity of field populations of R. solani AG-3 from potato and tobacco in North Carolina was examined using somatic incompatibility and amplified fragment length polymorphism (AFLP) criteria. A sample of 38 and 36 isolates of R. solani AG-3 from potato and tobacco, respectively, were paired in all possible combinations on potato dextrose agar containing 2% activated charcoal and examined for their resulting somatic interactions. Pairings were also conducted and examined on glass slides. Thirty-four somatic compatibility groups were identified in the potato sample, while eight somatic compatibility groups were present in the tobacco sample. Thirty-eight and 26 AFLP patterns (using two different primer sets) were identified from the potato and tobacco samples, respectively. Five clones (i.e. cases where 2 or more isolates shared the same AFLP pattern and were somatically compatible) were identified in the tobacco sample, while no clones were present in the potato sample. Compatible interactions only occurred between potato isolates with similar, but not the same AFLP pattern, from the same field (two isolates in each of four different fields). None of the potato or tobacco isolates were compatible or shared a common AFLP pattern.
Department of Botany, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C.
Rhizoctonia solani Kühn is a worldwide soil-born pathogenic fungus, which can cause damping-off, stem rot, root rot, and leaf-spot. It shows a tremendous variation in characteristics such as geographic location, morphology, host specificity and pathogenicity. Until now, the pathogenic gene has been unknown.
Numerous methods have been used to research the Rhizoctonia solani pathogenic gene(s), such as random amplified polymorphic DNA RAPD), ITS sequences, linear form plasmid etc. In this study, we chose nine local, different virulent isolates belonging to the anastomosis group 4 (AG4) and a standard isolate from Japan belonging to AG4 and tested the cellulase activity. Using a pair of primers designed from Phanerochaete chrysosporium cbh-1, Neurospora crassa cbh-1, Pencilium janthinellum cbh-1 and polymerase chain reaction, the exo-cellulase gene of Rhizoctonia solani was cloned into the pGEM3Zf+ plasmid and sequenced. According to the results, the relationship between the virulence, pathogenicity and variation in the exo-cellulase gene was determined.