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.

Molecular phylogeny of Ceratobasidium spp. (BNR) based on rDNA-ITS sequences

V. Rubio¹, V. González¹, M. de los Angeles Portal¹, J. Acero¹, O. Salazar¹, M. del Carmen Julián¹, M. Hyakumachi² and B. Sneh³

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.

Seamless cellulose tubing for the observation of anastomosis of Rhizoctonia solani

S.P.Y. Hsieh and R.Z. Huang

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.

Anastomosis grouping of Rhizoctonia solani isolates from turfgrass based on ITS sequencing

T. Hsiang

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.

Exploration of pathogenicity and DNA variation of Rhizoctonia solani AG-4

P.T. Huang and J.S.M. Tschen

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.

Cytomorphological, molecular and pathogenic characterization of Rhizoctonia solani associated with soybean from Brazil

R.C. Fenille^1,2, E.E. Kuramae¹, N.L. Souza¹

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.

Using pectic zymogram as a criterion to study variation of pathogenecity of field isolates of Rhizoctonia solani

G.R. Balali, A.M. Kasalkheh and M. Kowsari

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.

Phylogeny of anastomosis groups of Rhizoctonia solani based on sequence analyses of LSU and ITS regions of nuclear ribosomal DNA

D. González¹, M.A. Cubeta² and R. Vilgalys³

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.

Comparison of isolates of Rhizoctonia solani AG-2 and AG-BI based on anastomosis reactions, rDNA sequence analysis and pathogenic potential

S. Kuninaga ¹ and D.E. Carling ²

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.

Pathogenicity and anastomosis groups of Rhizoctonia isolated from potato tubers in Argentina

L. Casadei María¹, L. Gasoni¹ and M. Rivera²

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.

Rhizoctonia species pathogenic of rice in Argentina

M.A. Mazzanti de Castañón¹, S.A. Gutiérrez¹ and L.A. Gasoni²

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.

Isolates of Rhizoctonia solani HGII AG4 anastomosis group causing damping-off in seedlings of Coffea arabica in Brazil

E.E. Kuramae^1,2, A.A.B. Sussel¹, R.C. Fenille¹, N.L. de Souza¹

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.

Distribution of anastomosis groups of Rhizoctonia solani on potato in Mexico

G. Virgen-Calleros¹, V. Olalde-Portugal², S. Gomez-Sumuano², R. Hernandez- Matehuala² and D. Carling³

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.

Characterization of Rhizoctonia solani occurring in sugar beet in the Netherlands

J.H.M. Schneider

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.

The anastomosis groups of Rhizoctonia solani Kuhn from Taiwan

T.F. Hsieh¹, Y.C. Chang¹, and C.C. Tu²

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.}

Occurence of anastomosis groups of Rhizoctonia spp. isolated from different plants in Turkey

G. Tuncer¹ and G. Erdiller²

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.

Towards the development of a molecular identification system of Rhizoctonia solani strains: Comparison of AFLP patterns and ITS sequences with anastomosis grouping

E.C.A. Abeln, D. van Gosliga, O. de Vries, J. Steenbergen, P.H.J.F. van den Boogert, P. Bonants, IJ. Vlug and J.A. Stalpers

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.

Characterization of anastomosis groups and pathogenicity of Rhizoctonia solani of lettuce and tomato in Brazil

E.E. Kuramae^1,2, A.L. Buzeto¹ and N.L. de Souza¹

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.

Session 2: Population Biology and Genetics

Development of a PCR-RFLP method for examining the genetic diversity and structure in populations of Rhizoctonia solani AG-3 from potato and tobacco in eastern North Carolina

P.C. Ceresini^1,2, H.D. Shew¹, R.Vilgalys³, U.L. Rosewich⁴ and M.A. Cubeta⁵

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.

Population biology and genetics of Rhizoctonia solani anastomosis group 3

M.A. Cubeta¹, P.C. Ceresini^2,3, H.D. Shew² and R.Vilgalys⁴

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.

Structural and functional analysis of hairpin loop DNA plasmid from Rhizoctonia solani

T. Hashiba, A. Sasaki, K. Katsura, and A. Nagasaka

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.

Characterisation, pathogenicity and genetic variation in isolates of Rhizoctonia solani AG-3 from potato

A.J. Hilton, S.L. Linton and A.K. Lees

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.

Genetic diversity of populations of Rhizoctonia solani AG 2-2-IV associated with single basidiospore isolates of Thanatephorus cucumeris in a sugarbeet field in Japan

S. Naito

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.

Molecular and genetic characterisation of Australian and overseas Rhizoctonia solani anastomosis group 2

B.J. Stodart^1,2, S.M. Neate³, E.S. Scott¹, K. Ophel-Keller², P.R. Harvey³ and D.L. Melanson⁴

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.

RAPD-PCR used to support concept of sub-populations within Rhizoctonia solani AG-8

G.C. MacNish and P.A. O’Brien

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.

Seedling blight of calla lily caused by Rhizoctonia solani in Taiwan

C.W. Chen¹, M.G. Chiu² and L.C. Chen²

C.W. Chen¹, M.G. Chiu² R.W. Lin² 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.

The diversity of the rice sheath blight pathogens in Taiwan

C.H. Chang, Y.Z. Lin, Y.M. Chang, F.L. Liu and L.C. Chen

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.

Genetic relationship among isolates of the web blight pathogen of common beans based on PCR-RFLP and sequence of the ITS-DNA region

G. Godoy-Lutz, J. R. Steadman, K. Powers and B. Higgings

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.

Phylogenetic relationships of Rhizoctonia solani AG4 based on ITS ribosomal DNA sequences.

V.L. Wang and J.S.M. Tschen

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.

rDNA nucleotide sequence analysis of Rhizoctonia solani associated with foliar blight on soybean in Brazil

R.C. Fenille^1,2, E. E. Kuramae¹, D. N. Nozaki¹and N.L. Souza¹

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.

Nuclear and mitochondrial rDNA sequences analysis of multinucleate and binucleate isolates of bean, peanut and AG testers of R. solani and Rhizoctonia spp. binucleate

E.E. Kuramae^1,2, D.N. Nozaki¹, R.C. Fenille¹, N.L. de Souza¹

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.

Function of mitochondirial hairpin loop DNA plasmid pRS224 from Rhizoctonia solani AG2-2-IV

A. Nagasaka, A. Sasaki, K. Katsura, T. Sasaki, and T. Hashiba

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.

Linear DNA plasmids in Taiwan Rhizoctonia solani AG4 isolates

Y.H. Tseng, H.C.F. Chow, M.J. Cheu, and J.S.M. Tschen

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.

The pathogencity of Rhizoctonia solani AG-4 related to genetic recombination, ITS region variation and plasmid presence

I.C. Ho, K.Y. Yü, J.N. Lin and J.S.M. Tschen

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.

Widespread distribution of plasmid-like DNAs of Rhizoctonia solani AG-4 isolates in Taiwan

J.T. Liao, Y.W. Chung J.F. Huang and L.C. Chen

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.

Pathogenic and genetic variation in Rhizoctonia bataticola (Macrophomina phaseolina) and resistance to the fungus in Phaseolus vulgaris L.¹

N. Mayek-Pérez², C. López-Castañeda³, R. Garcia-Espinossa³, M.M. González- Chavira⁴, J.A. Acosta-Gallegos⁴, O. Martínez de la Vega⁵ and J.W. Simpson⁵

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.

Somatic incompatibility and AFLP analyses indicate differences in population structure of Rhizoctonia solani AG-3 associated with potato and tobacco in North Carolina

P.C. Ceresini^1,2, H.D. Shew¹, R.Vilgalys³ and M.A. Cubeta⁴

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.

Cloning and characterization of the Rhizoctonia solani AG4 exo-cellulase gene

S.W. Tung and J.S.M. Tschen

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.

Session 3: Symbiosis and Plant Fungus Interactions

Symbiotic abilities of mycorrhizae isolated from terrestrially grown and epiphytic orchids

S. Narmatha Lekshmi, J.Y. Wong, T.K. Tan and C.S. Loh

Department of Biological Sciences, National University of Singapore, Lower Kent Ridge Road, Singapore - 117 600.

The roots of five terrestrially grown orchids and eleven epiphytic orchids were used to isolate orchidaceous mycorrhizal fungi. A number of other fungal isolates were also recovered. Significantly larger fungal counts and types of fungi were obtained from orchids with thick velamenous roots (Arachnis Maggie Oei and Vanda Miss Joaquim) than those with thin velamenous roots (A. graminifolia, S. plicata, B.finlaysoniana). However, the epiphytic orchids did not show any significance in this regard. Particular attention was paid to the symbiotic interactions of the six most frequently recovered Rhizoctonia isolates, Nq (Diplocaulobium enosmum), SP1a (Spathoglottis plicata), AM9 (Arachnis Maggie Oei), AG3a (A. graminifolia), VJ15 (Vanda Miss Joaquim), and Ss (Dendrobium crumenatum) with Dendrobium crumenatum and Spathoglottis plicata hosts. The most effective of these resulted in an average of 98% germination for D. crumenatum seeds and 75% germination for S. plicata seeds. Nq (recovered from an epiphytic D. enosmum) was the best isolate for D. crumenatum seeds, promoting their growth (average embryo length of 1036mm) and formation of leaves. Similarly, S. plicata seedlings with leaves and roots were obtained within the 8^th week of inoculation with the fungal isolates, with SP1a as the best isolate in promoting their growth and seedling development. Although the performance of the Nq and SP1a isolates suggested certain degree of specificity between the orchids and their mycorrhizal fungi, the infrequently recovered Ss isolate (from D. crumenatum) did not enhance growth and development of its D. crumenatum seeds but just remained generally symbiotic.

Effects of mycorrhizal infection on different stages of tissue-cultured Dendrobium sonia

S. Narmatha Lekshmi, T.K. Tan and C.S. Loh

Department of Biological Sciences, National University of Singapore, Lower Kent Ridge Road, Singapore 117 600.

Seed-derived Dendrobium Sonia tissues cultured in Knudson C medium were used. Callus tissues, protocorm-like bodies (PLB), rootless PLB at two-leaf stage and PLB with two leaves and a developing root, were inoculated stagewise with Rhizoctonia isolates SP1a, AM9, Nq and Ss and their subsequent development studied over 24 days. There was remarkable increase in the average percentage of PLBs (91.5%) infected with mycorrhizal fungus developing on to the next growth stage. This was significantly higher than that of the control (44.5%) and progressed at a faster rate than the infection at any other growth stage. The mycorrhizal infected tissues showed marked tissue differentiation within the first week whereas the control on MS media seemed to merely propagate its tissues, thus delaying its development into the next stage. Strain Ss, although compatible when infected at the initial growth stages, showed minimum growth (9.5%) with PLBs with two leafs, and caused soft rot in the later part of the study. This could be referred to as ‘breakaway parasitism’ (Hadley, 1970) in which the fungus changes its behaviour and ramifies through the tissues. A sequencial study on the above symbiotic infection at weekly intervals is in progress. Microtomed infected tissues are studied and peloton counts are made inorder to establish any correlation between the number of pelotons and the growth stimulus at the four tissue culture stages.

Is melanin biosynthesis essential for pathogenicity of Rhizoctonia spp.?

B. Sneh¹, and V. Rubio²

¹Department of Plant Sciences and Institute for nature Conservation Research, Tel Aviv University, Ramat Aviv, Israel 69978; ²Centro Nacional de Biotecnologia (CSIC-UAM). Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain

Generally, melanins are considered to confer tolerance to fungal hyphae, Sclerotia and spores against stressed environmental conditions, such as: UV irradiation, extreme temperatures, and desiccation, microbial lysis and plant - and animal - defense responses to fungal infections (Bell and Wheeler, 1986; Henson et al., 1999)

Although melanins are the most common fungal pigments, their synthetic pathways are not uniform. In the Ascomycotina and related Deuteromycotina, the melanins in cell walls are generally synthesized from acetate by the pentaketide pathway, in which 1,8-dihydroxynaphthalene (DHN) is the immediate precursor of the polymer. Another biosynthetic pathway for melanin in various fungi and other microorganisms is from tyrosine via 3,4-dihydroxyphenylalanine (DOPA). In the Basidiomycotina the melanins are derived from g-glutaminyl-3,4- dihidroxybenzene (GDHB) or cathecol, as the immediate phenolic precursor for melanin. The latter pathway was described for Agaricus bisporus (Bell and Wheeler, 1986; Henson et al., 1999).

In several plant pathogens (Pyricularia oryzae, Colletotrichum lagenarium and others) melanin deposition in their infection cushions has been confirmed to be essential for infection peg penetration into the plant cell wall. Deposition of melanin in the cell wall of the appressoria reduces cell permeability, which increases cell osmolite concentration and consequently increases cell turgor. Melanin also provides cell wall rigidity. These attributes provide the hyphal tips or infection pegs the capability to penetrate the host’s cell wall. It has been confirmed with the above mentioned fungi that have lost their pathogenicity by a mutation in one or more of the genes coding for enzymes involved in the melanin biosynthetic pathway. The pathogenicity was successfully restored by complementation of the defected genes. In Gaeumannomyces graminis f. sp. tritici, melanin formation has also been implicated in pathogenicity (Bell and Wheeler, 1986; Kubo et al., 2000).

Certain non-pathogenic isolates of Rhizoctonia spp. (np-R) have been shown to be highly effective in protecting plants against pathogenic Rhizoctonia spp. (Sneh, 1996) These non pathogenic isolates colonize the plants' outer surfaces with a dense layer of hyphal mats, but the hyphae do not penetrate the plant host cell wall. The hyphae of np-R isolates are usually hyaline, in contrast to the brownish color of pathogenic isolates (due to melanin deposition in their cell walls).

Confirming the genetic and biochemical basis of the pathogenicity in Rhizoctonia are of essential importance for the basic knowledge on the pathogenicity of this important group of pathogens and for improving alternative methods for Rhizoctonia disease control. The lack of melanin synthesis in the np-R isolates due to possible defects in certain genes involved in the melanin biosynthetic pathway may be one of the reasons for avirulence of non-pathogenic Rhizoctonia isolates

Pairing of certain non-pathogenic binucleate Rhizoctonia isolates on PDA plates resulted in formation of brown pigmentation in the region of contact of the isolates. In certain cases the pigmentation was spread to the entire colony of one isolate. Transfer of hyphae from the contact region to new plates resulted in non pigmented colonies. Further studies are needed to determine the mechanism of pigmentation in the contact region. Following are some possibilities: Complementation of enzymes in the synthetic pathway of melanin, Stress imposed in the contact region, which induced certain genes in the pathway, which are silent.

Further work is also required to find out whether the formation of melanin in the contact region renders the non-pathogenic isolates pathogenic. If the pigmentation in the contact region is only in he surrounding medium (due to secretion of phenol oxidases, which polymerize phenolic compounds present in the medium) and not in the cell wall it may not affect pathogenesis at all. On the other hand, other genes products are required n addition to melanin synthesis for pathogenesis which are responsible for: a) formation of enzymes involved in the process of (such as pectinases, cellulases, cutinases proteases etc.), b) specific host recognition, c) acting against host defense mechanisms or other additional qualifications.

The knowledge regarding the genes determining essential, as well as host-specific pathogenicity of Rhizoctonia spp. is rather poor. The obstacles, which need to be developed are: a reliable transformation system, and a reliable method to form the teleomorph stage for obtaining single basidiospore cultures.

Influence of orchid mycorrhizal fungi Rhizoctonia spp. on the growth of Oncidium Goldiana X Onc. Guiena Gold seedlings

C.L. Wang¹, J.N. Chu², C.Y. Chen¹ and Y. C. Chao³

¹Dept. of Plant Industry, National Pingtung University of Science and Technology (NPUST), Pingtung, Taiwan, R.O.C.; ²Institute of Tropical Agriculture, NPUST; ³Dept. of Plant Protection, NPUST

Soil beneficial fungi colonize the roots of higher plants to form a symbiotic associate structure named mycorrhizae. They are common found in the roots of numerous earth plants, and able to improve the growth of host plants. Some of the fungi symbiosis with orchid plants are called orchid mycorrhizal fungi, mostly belonging to the genus of Rhizoctonia, can enhance the germination of orchid seeds and improve the growth of seedlings. However, some of these orchid mycorrhizal fungi may become the plant pathogens in special environmental conditions. Oncidium spp. is one of the important economic flowers for export and domestic sale in Taiwan. The growing period from seedling to the mature plant is quite long. For the purpose of improving seedling growth, the beneficial mycorrhizal fungi were isolated from the roots of wild terrestrial and cultivated orchid plants, and inoculated on the roots of Oncidium Goldiana X Onc. Guiena Gold, to screen the most beneficial orchid mycorrhizal fungi.

In this experiment 23 isolates were acquired, two of them belong to Rhizoctonia. The isolate OS was derived from the roots of cultivar Oncidium lancy, and TF was acquired from the roots of wild Thrixspermum formosanum. Both isolates were inoculated to the micropropagated plantlets respectively, to investigate their effect on the growth of Oncidium sp. After 4 weeks, the results showed that OS and TF isolates improve the growth of plant height, length and width of mother and daughter pseudobulbs, shoot and root fresh weights and daughter pseudobulb number of orchid plants. It is obvious that OS and TF isolates have positive effect on the growth of Onicidium sp. Plant.

The pathogenicity test was conducted with OS and TF isolates, and Rhizoctonia solani AG-1 was serving as the control strain, they were inoculated to the seeds of mung bean, radish and paddy rice to investigate the disease index. The results showed that the disease index of control strain Rhizoctonia solani AG-1 reached 100%, 100% and 48.90% in mung bean, radish and paddy rice, while those for OS were 11.67%, 9.17% and 7.92%, 0%, 5.00% and 4.86% for TF. It was indicated in this experiment that isolated orchid mycorrhizal isolates, OS and TF, have fairly low pathogenicity to the three above-mentioned host plants. They have very high potential to be exploited a microbiofertilizer.

Rhizoctonia-like fungi associated terrestrial orchids in Thailand

L. Manoch¹, P. Athipunyakom² and M. Tanticharoen³

¹Department of Plant Pathology, Faculty of Agriculture, Kasetsart University, Bangkok 10900, Thailand; ²Department of Agriculture, Plant Pathologly and Microbiology Division, Bangkok 10900, Thailand; ³National Center for Genetic Engineering and Biotechnology (BIOTEC), Bangkok 10400, Thailand

The aim of this research was to isolate, cultivate, and identify Rhizoctonia species associated with the roots of terrestrial orchids. Five terrestrial orchids, namely Goodyera procera Paphiopedilum concolar, Paphiopedilum concolor var. striatum, Paphiopedilum sp., and Spathoglottis plicata were collected from various locations in Thailand, i.e. Chanthaburi, Kanchanaburi, Chiangmai and Bangkok. A direct isolation method was used by dissecting longitudinal orchid root sections and observing hyphal coils (pelotons) with stereo microscope in a clean bench. A fine needle was used to pick up a peloton and place it on Matsuhara & Katsuya NDY medium in a petridish. After 3 to 5 days incubation, hyphal tips were transferred onto PDA agar slants and kept as pure cultures for identification. Soil samples adjacent to orchid roots were also collected and fungi were isolated using the direct inoculation method onto cornmeal agar and the rice baiting technique.

A least seventy-five Rhizoctonia-like fungi were isolated. Among them, seventy isolates were derived from pelotons in the root tissues and the rest were found using methods. Some strains produced monilioid cells together with upright hyphae. The mycelium colors varied from light to dark brown, depending on the isolates. The morphological characteristics of all isolates were observed and recorded using a light microscope with Nomaraski Interference contrast and camera lucida drawings. Macroscopic characteristics were observed on artificial media at different temperatures. Comparisons between the different isolates of Rhizoctonia species from pelotons in root tissues and from soil samples were made. Nuclei were stained with safranin O using Bandoni𠏋 method. Most isolates were binucleate Rhizoctonia spp.

An over view of the host-pathogen interaction in damping-off of groundnut caused by Rhizoctonia solani and the disease cycle

M.N. Reddy

Department of Applied Microbiology, S. P. Mahila Visvavidyalayam, Tirupati-517502, India

Damping–off of groundnut (Arachis hypogaea L.), caused by Rhizoctonia solani Kuhn, is a seeding disease resulting in total death of the seedling, by the development of water-soaked lesions on hypocotyls, which later turn brown, dark brown and sunken. The disease progressed in five fairly distinct stages of lesion development and maturation. The behavior of the pathogen on the surface of the host, in relation to infection cushion formation and disease development was studied and observed gradual reduction in the disease intensity and ultimate development of resistance to infection by the time the seedling attains the age of 21 days. Changes in the exudation pattern, anatomy of the seedling hypocotyls, with increasing age of the seedling in relation to disease occurrence and development of resistance were also studied. The tissues, both infected and the corresponding healthy, at different stages of disease development were sampled and analysed for different physiological and biochemical constituents and observed significant and also very characteristic dysfunctioning, of both qualitative and quantitative nature, as a result of infection. Biochemical characteristics of the pathogen were also studied to understand the role of the pathogen in pathogenesis. A definite correlation was evident between the observed alterations, development of disease, visual symptoms and consequent death of seedling. At least in part, these changes are mediated through the activity of the pathogen or host-pathogen interaction or both. The significance of the results is discussed in the light of the available literature on host-parasite relations. By taking into account the results of various aspects, of the pathogen, the host, the disease and the host-pathogen interaction investigated and those that were not covered but well established, a scheme has been derived to visualize sequential steps and mechanism(s) underlying in damping-off of groundnut.

Session 4: Ecology and Epidemiology

Epidemiology of rice sheath blight caused by Rhizoctonia solani Kühn under tropical conditions

N. Castilla¹ and S. Savary²

¹Entomology and Plant Pathology Division, International Rice Research Institute, MCPO Box 3127, 1271 Makati City, Philippines; ²Institut de Recherche pour le Développement, 911 avenue Agropolis, BP 5045, F - 34032 Montpellier cedex 1, France

The spatio-temporal dynamics of spontaneous epidemics of rice sheath blight caused by Rhizoctonia solani Kühn were studied in two consecutive cropping seasons in a farmer's field in the Philippines. The rainy season epidemic had a lower primary inoculum and a higher rate of disease increase, whereas the dry season epidemic had a higher primary inoculum and lower rate of disease increase. Semivariograms indicated that disease aggregation coincided with the phases when the rate of disease increase was high. Spatio-temporal autocorrelation analysis at 45 days after transplanting showed that disease incidence of a rice hill was correlated only with that of its nearest sampled neighbors in the rainy season. No spatial correlation was found in the dry season epidemic. Another set of experiments showed that aerial, leaf-to-leaf spread was greater in denser stands, optimum under alternate wet and dry periods, and decreased with nitrogen content of the plants. Nitrogen fertilizer indirectly favored the disease by increasing contact frequency and leaf wetness in the canopy. Further experiments showed that flooding the soil for two weeks reduced the efficiency of soil inoculum of rice sheath blight. Flooding also reduced the efficiency of colonized rice stems and rice grain/hull mixture but did not affect the efficiency of sclerotia. These studies show that the leafborne phase of rice sheath blight epidemics should be targeted to control the disease because it has a stronger effect on the rate of disease increase than does the soilborne phase.

Effect of spatial pattern of initial infections on the epidemics of rice sheath blight caused by Rhizoctonia solani Kuhn

N. Castilla¹ and S. Savary²

The effect of the spatial pattern of initial infections on the epidemics of rice sheath blight caused by Rhizoctonia solani Kuhn was studied during two consecutive rainy seasons. We report here experiments involving four treatments (three different spatial patterns of initial infections and a non-inoculated control) which were established in microplots with 15 x 15 hills each. The random pattern was established by randomly inoculating nine hills, the uniform pattern by inoculating every other four hills, and the aggregate pattern by inoculating nine central hills. In 1995, disease incidence was higher in treatments with random and uniform patterns than in treatments with aggregated pattern and the control at 14 to 35 days after inoculation (DAI). In 1996, disease incidence in random and uniform pattern treatments was significantly higher than in the aggregated pattern treatment and the control at 10 to 28 DAI. At 31 DAI, the uniform pattern treatment had the significantly highest disease incidence. The ranking in decreasing order of the area under disease incidence progress curve (AUDIPC) in both years was random, uniform, aggregated, and control treatments. These results show the importance of the degree of aggregation of initial infections on

the leafborne phase of the rice sheath blight epidemic. Considering that interplant disease spread depends on contact frequency, diseased hills in plots with uniform and random spatial patterns of initial infections provide greater accessibility to healthy hills to the pathogen, resulting in a higher number of infected hills than in plots with aggregated pattern of initial infections.

Three seedling diseases of bitter gourd-loofah graft and their control

Y.S. Lin, K.S. Huang and C.H. Hwang

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, R.O.C.

Fusarium wilt of bitter gourd could be controlled by using bitter gourd shoot as scion cleft-grafted on loofah as rootstock, but it was not practical in fields due to three new limiting seedling diseases. In winter, seedling blight caused by Alternaria alternata f. sp. cucurbitae attacked the grafted seedlings at 8-16℃. Sunscald and damping off of grafted seedlings occurred severely in nursery bed in the summer time. The sunscald occurred when the temperature was above 28℃. The damping off caused by Rhizoctonia solani AG-4 was prevalent when the environment was humid and above 24℃, with inoculum originating from peat moss used as cultural substrate. Spraying of flutolanil, pencycuron and polyoxin B at 500 ppm could reduce the damping off to a certain extent. This study was proposed to develop the non-chemical methods which are effective against all the three seedling diseases. Using clean seeds and cultural substrate, employing bitter gourd seedling from seed, instead of shoot, as scion to be cleft-grafted or cleft-inarched on loofah to produce graft seedlings growing at 20℃ are the selected methods highly recommended. The growth of the grafts in the field several years has resulted in gradual disappearance of Fusarium wilt and the better yields of bitter gourd reached 80-100 t/ha, as compared to the average yield of 16 t/ha for the non-grafted bitter gourd in Taiwan.

Web blight of Urdbean and Mung bean in North India

R.P. Dwivedi

Department of Plant Pathology, C. S. Azad University of Agriculture & Technology, Kanpur-208 002 (UP) INDIA

Urdbean (Vigna mungo) and Mungbean (Vigna radiata) the important pulse crops of India, suffer from a destructive web blight disease caused by Thanatephorus cucumeris (Fr.) Donk (=Rhizoctonia solani Kühn), AG-1, causing considerable losses every year in northern part of India. Disease intensity gradually increased in the field and reached maximum during second fortnight of September in temperature range of 22-30ºC and relative humidity of 70-95 per cent. Under warm and humid conditions with persistent overcastted sky and continuous rains providing maximum dew formation on the leaves favour the inoculum build up and disease development and spread rapidly to cover large plant population. Maximum disease development occurred at temperatures between 26-28ºC and relative humidity 90-100 per cent. It also influenced the perfect state development in nature. Thirty days old plants were highly susceptible. Infected seeds, plant debris, collateral hosts and sclerotia served as primary inoculum where as secondary spread occur through basiodiospores and contact between diseased and healthy plant parts.

In laboratory and field experiments flexaconazole, carbendazim, isoprobenphos, thiophanate methyl, cholorothalonil, mancozeb and captan were found effective in reducing radial growth and sclerotial production of pathogen and disease intensity.Cost effective integrated management was found through seed treatment with carbendazim, 2gm/kg seed followed by spraying of carbendazim (0,05%) or thiophanate methyl (0.05%) or flexaconazole (0.05%) in both the crops.

Soil application or seed treatment with Gliocladium virens alone or in combination with Thiram or Captan increased the seed germination and decreased the disease development.

The occurrence situation of rice sheath blight in Taiwan and pathogen survival tests

C.H. Chang, Y.Z. Lin, Z.R. Chang and L.C. Chen

Dept. of Plant Pathology, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, ROC

We paid visits to many rustic villages, outskirts of cities, and some agricultural research organizations. To do the planting of rice is a low-income occupation at present in Taiwan. It's an income-earning sideline of most rice-planting farmers. It limits the occurrence of rice sheath blight in partial areas that the cultivated land has been divided up, changed in the kinds of crops, or deviated for constructing buildings, and the pesticides have been used excessively. The rice fields are rarely seen in the major cities of industry and commerce. We are of the opinion that the rice sheath blight at present in Taiwan is still serious as in 20 years and become to be the most important diseases except rice blasts. Under screened by the environmental pressures, we believe that maybe the survival ability of the remained isolates is higher than that in 1981. (Tu & Chang, 1981) At that time the survival percentage of sclerotia of Rhizoctonia solani AG-1 IA in 14-cm depth of the flooding soil is 6% after 2 month. So we collected more than one hundred isolates from many areas and tested the survival ability as well as proved the inevitabile inclination of biographical evolution.

Anastomosis groups of Rhizoctonia solani isolates from potatoes in Pakistan

C. Abdul Rauf¹, M. Ashraf² and I. Ahmad³

¹Department of Plant Pathology, University of Arid Agriculture, Rawalpindi; ²Department of Biological Sciences, Quaid-i-Azam University, Islamabad; ³Crop Disease Research Institute, National Agriculture Research Centre, Islamabad

Potato black scurf caused by Rhizoctonia solani Kuhn is a common fungal disease in six different potato production agro-ecological areas (Sahiwal, Attock, Swat, Kalam Valley,Chitral and Quetta) of Pakistan. For anastomosis (hyphal fusion) grouping, forty-six isolates of R. solani Kuhn obtained from sclerotia bearing potato tubers grown in different potato production agro-ecological areas were tested against eight known tester isolates viz., AG 1-1A, AG B1, AG 1-1C, AG 3, AG 4, AG 6, AG 7 and AG 8 of R. solani. All isolates tested in this study were multinucleate. Anastomosis grouping revealed that 45.67% of the isolates belonged to anastomosis group 3 (AG 3), and the remaining were member of Ag 1-1A , AG 6, AG 4, AG 7 and AG 2-1 which accounted for 8.69%, 6.52%, 4.34% and 2.17% respectively. None of the isolate anastomosed with AG B1 and AG 1-1C tester isolates. The Percentage of isolates which did not anastomose with any of the eight tester isolates was 23.91%. This is first report of anastomosis grouping in R. solani from Pakistan.

Population dynamics and survival of Rhizoctonia solani AG-1 in field soil

S. Li

Biological Control Institute, The Chinese Academy of Agricultural Sciences, Beijing 100081

Selerotia, colonized plant debris and free hypha of Rhizoctonia solani AG-1 was monitored quantitatively in a rice-wheat rotation field near Chengdu for one year. Inoculum density peaked in March and September, with 1222 and 480 colonized debris 100g^-1 air dry soil, 7.9mg and 1209mg fresh wt mycelium g^-1 air dry soil, and no detectable sclerotia, respectively. And the inoculum density valleyed in December and August, with 3 and 289 colonized debris 100g^-1 air dry soil, 3 and 0 sclerotia 100g^-1 air dry soil, and no detectable mycelium at each time. Colonized plant debris was the dominant inoculum type of R.solani AG-1in soil in most of time around the year. However only sclerotia and large colonized plant debris (>2.00mm in size) could survive over winter, albeit their population were low at that period. Free hyphae were only detected in March, April and September, not in other time. Soil temperature was considered as an important factor affecting the fluctuation of R.solani AG-1 population in natural soil. The suitable soil temperature range for building-up of the fungus population was assumed from 13C to 22C. Viability tests indicated that 60.9% sclerotia could survive at 265 days after burying in natural sandy loam under field conditions, while colonized rice debris (0.5-1.0cm in length) could not yield the fungus any more at 88 days after the burying.

Non target effect of agro chemicals on Rhizoctonia sp.

S. Livingston

c/o Dr. (Mrs) Pramila Gupta, Prof & Head, Dept of Plant Pathology & Nematology, Allahabad Agri. Institute, Allahabad, U.P. India

The use of chemicals to prevent plant diseases started with the discovery of Bordeaux mixture in 1885 by Millardet. Until biological control methods are shown to be practical on large scale, the increase use of pesticides can be fully justified. These agro chemicals applied to soil effects not only the target organisms but also the non target organisms. It may be of interest and immense importance to determine if there are any relationship between the increase and decrease of diseases in chemically treated crops. Soil borne fungus Rhizoctonia sp. causes damping off, wilt, root rot in many economically important crops causing considerable reduction in yield. It is paramount importance to find out before recommending their applications in the fields, the way these chemicals act on non target microorganisms. With this in view the present studies were carried out.

Rhizoctonia sp. was isolated from infected plant materials and subcultured till pure cultures were available. Various agrochemicals namely, insecticides, nematicides, fungicides, herbicides were used in the study. The chemicals were prepared in 10,000, 1000, 100 and 10 ppm concentrations and the effect of the chemicals were studied by Poison Food Technique in vitro. The radial growth and the weight of the mycelium were observed. In vivo studies, the isolated Rhizoctonia sp. was mixed with sterilized soil in pots and the chemicals were applied at the recommended method and dose. Observations were recorded on germination percent and disease ratings.

In vitro when the insecticides, nematicides, fungicides, herbicides were observed to inhibit the growth of fungus significantly in higher concentrations.

In vivo studies, observations were recorded on germination percentage and disease ratings. It was observed that in all the chemical treatments, the germination percent was significantly higher than control (55.5%) ranging from 77 % 100 % except for one herbicide were the germination was 11%. When the disease rating index was observed, it was found that the control plants were at maximum grade of 5%, where as the disease ratings were less than 2 % in all the treatments except for phorate treated plants where the disease ratings was 4%.

This study shows that the chemicals not only attack the targeted organisms but also the non target organisms.

Factors affecting formation of hymenia and basidiospores of Thanatephorus cucumeris ( teleomorph of Rhizoctonia solani AG-2-2 ⅢB ), the causal agent of Chinese amaranth leaf spot

J.W. Huang¹, H.F. Lin¹ and T.F. Hsieh²

¹Department of Plant pathology, National Chung Hsing University, Taichung, Taiwan ; ²Department of Plant pathology, Taiwan Agricultural Research Institute, COA, Executive Yuan, Taiwan, ROC.

A new leaf spot disease of Chinese amaranth (Amaranth mangotanus L.) caused by Rhizoctonia solani AG-2-2 ⅢB was frequently observed at the so-called organic farms in central Taiwan during the summer season. Symptoms consisted of circular, brown, necrotic leaf spots, and claw-like chlorotic borders around the lesions. According to distribution pattern of the leaf spots, it seemed that the basidiospores of Thanatephorus cucumeris were considered as to play an important role in infection process. Therefore, the objectives of this study were to investigate the biotic and abiotic factors affecting the formation of hymenia and basidiospores of the pathogen. The sporulation ability of T. cucumeris on soil surface was estimated by the modified soil-cover-agar method described by Naito, et. al., 1995. Nine factors affecting formation of hymenia and basidiospores of the pathogen were conducted involving the isolates of the fungus, components of cultural media, soil textures, temperatures, pH values, organic and inorganic amendments, soil microorganisms and fungicides. Eight isolates of the pathogen collected from Hsilo, Taichung and Mauli areas showed significant difference between their sporulation ability. RSA-03, RSA-06, RSA-07 and RSA-09 isolates were able to produce more amounts of hymenia and basidiospores compared to the others. In addition, these isolates were markedly favorite to form hymenia at 26-28℃ on soils with pH values from six to eight. Among ten cultural media for growth of the pathogen studied, the addition of yeast extract to the media was essential for hymenia and basidiospores production. Twenty-four organic and inorganic amendments were added individually to loamy soil covering on a mycelial colony of T. cucumeris grown on potato yeast extract dextrose agar plate. Hymenial formation by the pathogen was significantly stimulated with increasing amounts of BVB NO.4 peat moss between 1 and 40%(v/v) in the soils. However, it was completely inhibited in the soils amended with 1﹪(v/v) tea seed pomace. The suppressiveness of soil amended respectively with seven fungicides and seven biocontrol agents to hymenial formation of T. cucumeris isolate RSA-09 was tested with the modified soil-cover-agar method. Results indicated that Mancozeb, Flutolanil, Bacillus subtilis BS-001, B. pumilus PMB-207, B. thermoglucosidasius PMB-102 and Streptomyces saraceticus SS-31 were more effective in inhibiting the fungus.

Significance of the blast disease (pythium splendens braun and Rhizoctonia lamellifera small) in oil palm cultivation in Nigeria

C.E. Airede

Plant Pathology Division, Nigeria Institute for Oil Palm Research (NIFOR), P. M. B. 1030, Benin City, Nigeria

Blast disease caused by Pythium splendens and Rhizoctonia lamellifera, is the most serious root disease affecting oil palm nursery seedlings throughout West Africa and in part of Malaysia. Seedling losses of up to 10-80% have been recorded. Virtually all seedlings affected by the disease, ultimately die and few that survive (25%) are malformed, stunted and generally unsuitable for field transplanting.

This paper reviews the research efforts made to date in the epidemiology and control of the disease in Nigeria. The significance of Rhizoctonia lamellifera in the complex eatiology of the disease is highlighted. R. lamellifera is not capable of penetrating healthy undamaged roots but establishes itself by first parasitising P. splendens, thereby, gaining access to the internal root tissues, Cultural studies showed that cultures of R lamellifera were pigmented. The best temperature range for growth, sponrangial and sclerotial production and germination was 25-30^oC and were better in agar media rich in simple sugars than in those deficient in them. Sclerotial longevity was positively correlated with relative humidity but negatively correlated with temperature and there was no loss of sclerotial viability at 20^oC while more than 50% of the sclerotia at 95% r. h. remained viable for more than one year.

Thee role of climatic factors and cultural practices in the eatiology of the disease is discussed. The most effective means of reducing the disease, is through a combination of cultural methods, including planting of sprouted seeds in the wet season in April or May into black polythene bags filled with air dried top soil, watering and lowering of soil temperature by mulching. The need for an integrated, cultural, biological and chemical practices for reducing losses is proposed.

Relationship between dispersal of rice sclerotial disease fungi, Rhizoctonia oryzae-sativae and R. oryzae, and the disease development in a paddy field, on the basis of survey of vegetative compatibility groups

A. Kamio and K. Inagaki

Faculty of Agriculture, Meijo University, Tenpaku, Nagoya 468-8502, Japan

In a field (15a), 48, 134, and 24 isolates of brown sclerotium disease fungus, R. oryzae-sativae, were obtained in 1998 - 1999 from lesions on the leaf sheaths of maturing rice plants (September), stubble and soils in the pretransplanting season (March), and weeds grown in and around the field in the rice tillering to maturing stage (June to Sepember), respectively. These isolates were classified into many vegetative compatibility groups (VCGs) that were determined by existence of barrage zone reaction at the colony interface when isolates were cultured face to face on the malt extract agar at 30 C for 7 -10 days. Four among 24 VCGs derived from 48 lesion isolates were common to 4 among 41 VCGs from 134 stubble/soil isolates, and 1 VCGs from lesions common to 1 among 6 VCGs from 24 weed isolates. In Rhizoctonia oryzae, causal fungus of rice bordered sheath spot, 3 among 5 VCGs from 8 lesion isolates were common to 3 among 11 VCGs from 64 stubble/ soil isolates, and 2 VCGs from lesions common to 2 among 11 VCGs from 27 weed isolates. These results indicate that in many cases R. oryzae-sativae / R. oryzae dispersing widespread in the field at the pretransplanting season arequantitatively andqualitaitvely different from those which cause sclerotial diseases about 6 month later in the same field. Weeds growing in and around fields proved to be conducive to survival of these pathogens.

Rhizoctonia in cereals in Venezuela

R. Cardona¹, H. Rodriguez¹, L. Cedeño² and H. Nass³.

¹FONAIAP-Portuguesa, Apdo. 102, Acarigua-Araure 3101-A, Portuguesa Venezuela; ²Instituto de Investigaciones Agropecuarias. Apdo. 77, Universidad de Los Andes, Mérida 5101-A Mérida, Venezuela; ³Estación Experimental Yaritagua (FONAIAP) Entrada Las Canarias Km 3 vía El Rodeo. Yaritagua, estado Yaracuy, Venezuela

It was confirmed that the fungus belonging to the anastomosis group AG-1-IA of Rhizoctonia solani Kuhn is the causal organism of the blight affecting stem, leaves and sheath on rice and maize where it also affects the ears. The characteristic syntoms are oblong to ellipsoid lesions, initially water-soaked and pale green in color. The lesion center turns white-gray and is surrounded by an irregular purple-brown border. In the isolates obtained, nuclear staining with HCl-Giemsa and observation under the electronic microscope showed that somatic cells were multinucleated. In distinct terminal and penultimate cells the number of nuclei varied between 3 and 7. The isolated fungus consistently anastomosed with the strain CBS 205.84 of the anastomosis group AG-1-IA acquired from the Centraalbureau voor Schimmelcultures, Netherlands. On PDA the fungus produced yellowish-brown colonies and brown semi-spherical sclerotia, not differentiated in medulla and cortex. The inoculations of healthy maize and rice plants produced symptoms and signs similar to those seen in the field. R. solani was consistently isolated from experimentally infected tissues. Since 1992 different fungicides have been probed for the control of R. solani in maize and rice fields.

Seedling blight of some cucurbitacious crops caused by Rhizoctonia solani

J.G. Tsay and R.S. Chen

Department of Plant Protection, National Chiayi University, Chiayi 600, Taiwan, (ROC)

In 1995, seedling blight of cucumbers was first described in Taiwan. The disease caused a reduction in seedling emergence and damping-off symptom. Root and crown lesions on young seedlings are pale brown. Many fungi associated with diseased root systems in vine decline symptoms of muskmelon have been reported in Taiwan. Pythium spp. was usually assumed to be the major pathogen which affected cucumbers. The pathogenicity of Rhizoctonia solani had been confirmed, but the host ranges of the pathogen within Cucurbitaceae had not been reported. Isolate RS-CH1 (AG1ⅠA) , and isolate RS-W3 (anastomosis group undecided) of R. solani were isolated from diseased roots of muskmelon and watermelon. Inoculums prepared from selected isolates were used to inoculate seedlings of melon (Cucumis melo), cucumber (Cucumis sativus), watermelon (Citrullus vulgaris), pumpkin (Cucurbita moschata), and calabash gourd (Lagenaria siceraria). Isolate RS-CH1 from muskmelon was more pathogenic than isolate RS-W3. Based on the results from AG1ⅠA, cultivars of melon were shown to be more susceptible to R. solani than other cucurbit species. Among tested melon cultivars, “Jade” and “Silver Light” were the most susceptible to R. solani. Isolate RS-W3, which inhibited seedling growth and showed no damping-off incited, was weakly pathogenic on tested cucurbits.

Development of the International Rhizoctonia Committee website

M.A. Cubeta¹ and S.M. Neate²

¹North Carolina State University, Department of Plant Pathology, 207 Research Station Road, Plymouth, NC 27962; ²CSIRO Division of Soils, Adelaide, South Australia

The International Rhizoctonia Committee (IRC) of the International Society of Plant Pathology has established a website (http://plymouth.ces.state.nc.us/isr/) to provide background information about IRC members Marc Cubeta (Newsletter and Website Editor), Gerda Dijst (Secretary/Treasurer), Suha Jabaiji-Hare (Vice-Chair), Shigeo Naito (Coordinator of Eastern Countries), Stephen Neate (Chair), Hans Schneider (Coordinator of Taxonomy Committee), and Baruch Sneh (Immediate Past Chair); including the establishment, history and objectives of the IRC. The website also has a newsletter with additional information about employment opportunities, links to other Rhizoctonia websites and resources, recent publications and a schedule of upcoming scientific meetings. The purpose of the website is to promote the dissemination of accurate and useful Rhizoctonia-related information (particularly current research updates) in a timely manner with the hope of stimulating discussion and exchange of ideas. The website is in a constant state of development and input is often needed from the Rhizoctonia community. Participants involved with the International Rhizoctonia Symposium in Taiwan are invited to provide comments and suggestions for improving the website content, format and quality to meet the needs of the Rhizoctonia community in the future.

Interaction effect of Rhizobium and Rhizoctonia solani on growth and yield of cowpea in black and red soils

R.R. Mallikarjunaiah and M. Vasanthakrishna

Department of Microbiology, University of Agricultural Science, G.K.V.K, Bangalore 560065, India

Soil acts as a habitat for complex population of microorganisms and it is the basis for agriculture. Black and red soil fractions like montomorillonite, kaolinite and bentonite protected the gram negative, nitrogen-fixing bacteria from desiccation and these stimulated population have suffered the growth of soil borne plant pathogens like Rhizoctonia, Verticillium, Fusarium etc.

This may be due to the effect of soil fractions, on toxins and fungistatic substances, produced by the nitrogen fixers. The complexity and density of soil microorganisms implies many interactions among them. Cowpea is an important food legume in humid and semi-arid tropics. Protein content of seeds varies from 23-28% and hence it forms a good protein substitute for vegetarians. Cowpea is susceptible to various diseases of which “root rot”; caused by Rhizoctonia solani is a serious disease of this crop. Negative results from the use of chemicals have encouraged the biological scientists to concentrate on biological control of pathogens to plants.

The beneficial effect of Rhizobium is well known on crop production, this may be due to Nitrogen fixation, production of plant growth hormones and fungistatic substances.

The present study was conducted to examine the feasibility of beneficial interaction between Rhizobium and Rhizoctonia solani in black and red soils as regards to (1) Nitrogen fixation; (2) Disease control and; (3) Plant growth and yield.

Session 5: Diagnosis and Detection

Trials and tribulations of DNA-based detection of Rhizoctonia solani AG-8 in soil

D.L. Melanson¹, I. Dumitrescu² and S.M. Neate²

¹Oregon State University, Columbia Basin Agricultural Research Center, P.O. Box 370, Pendleton, Oregon 97801, USA; ²CSIRO Land and Water, Private Mail Bag No. 2, Glen Osmond, South Australia 5064, Australia

The development of molecular markers specific to Rhizoctonia solani AG-8, together with improved methods and commercial kits for the extraction of DNA from soil, have considerable potential for studies on the population dynamics of this fungus. While the new techniques should provide for accurate detection of this fungus in field samples, with increased sensitivity, quantification and rapid and high volume sampling, there are problems with reliability. A series of experiments have been carried out to resolve inconsistencies in the direct detection of AG-8 in soil samples using the specific marker, pRAG12, in a slot blot assay. The level of Rhizoctonia present in total DNA extracted from different amounts of soil using commercial kits and laboratory methods was quantified using a Phosphor Imager and compared. Methods to improve the sensitivity, quantification and reliability will be described as well as possibilities of using PCR. Sampling strategy, including size, number and time of year, and potential for false positives need to be considered if used as a diagnostic test. While the technical hurdles of DNA-based detection and quantification appear surmountable, the correlation between the amount of Rhizoctonia present in the soil and disease severity is an enigma, with disease expression strongly influenced by agronomic practices and environmental effects. Because of the patchy nature of the disease in the field, small quantities of fungus present in soil and plant matter and the diversity of soil chemistry, physics and microbiology, it is important to understand the population dynamics of Rhizoctonia in order to use molecular detection as a risk management tool.

Should aggressiveness or subgroup identity of Rhizoctonia solani be subject for developing detection and control tools?

P.H.J.F. van den Boogert, F.D. Soglio, and A. Luttikholt.

Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands

In most of the Rhizoctonia solani pathosystems, aggressiveness of the fungus biotype is an important characteristic, affecting disease incidence and severity and population dynamics, including competition, survival and dispersion of the fungus. A better understand of aggressiveness and subgroup identity of R. solani may help to formulate targets for developing detection tools and control strategies. Rhizoctonia solani field isolates, belonging to AG 2, sub-groups 2-1 and 2-2IIIB, with differences in aggressiveness to cauliflower and sugar beet have been used for studies on various aspects related to aggressiveness. Molecular detection and fingerprint methods (ITS-PCR, AFLP and pectin zymograms) for different subgroups and biotypes of R. solani are available at Plant Research International. In greenhouse experiments, the effect of differences in aggressiveness on the soil colonization, biotype selection and disease expression have been assessed with mixtures of isolates, after successive host plantings. Results of this experiment indicates that the determination of biotype aggressiveness is probably more important than subgroup identity. There was differential interaction between aggressiveness and temperature. Indications of competition or synergism between different biotypes were also found. Isolates that differ in aggressiveness in vivo did not presented such differences in vitro. We were not able to correlate variation in biotype aggressiveness to cytoplasmatic ds-RNA profiles.

With the tools developed, it is possible to further study the genetics of aggressiveness, its relation to the population ecology of R. solani, and the response to different control strategies.

A specific and quantitative assay for the detection of Rhizoctonia solani AG-3 from potato and soil

A.K. Lees, D.W. Cullen and A.J. Hilton

Unit of Mycology, Bacteriology and Nematology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

Rhizoctonia solani AG-3 causes stem canker and black scurf of potato. Stem canker symptoms include stolon pruning and stem lesions, and can result in reduced tuber yield, whilst black scurf is characterised by the formation of irregular sclerotia on the tuber surface and leads to loss of skin quality. Both seed and soil-borne inoculum can cause infection of potato by R. solani, although the relative importance of inoculum type is not well understood.

Further investigations into the ecology and epidemiology of the diseases of potato caused by R. solani are needed, and will be facilitated by the availability of a simple, specific and quantitative diagnostic assay.

In order to develop such an assay, a primer pair was generated from the nuclear ribosomal internal transcribed spacer (ITS) region of R. solani AG-3. These primers were found to amplify DNA of AG-3 isolates, resulting in a 0.5 kb amplification product. However, DNA extracted from other R. solani anastomosis groups or other soil-borne fungi was not amplified. The assay was also able to detect AG-3 DNA from contaminated soil and potato tissue. In addition, a TaqMan fluorogenic probe was designed, allowing real-time quantitative (TaqMan™) PCR to be carried out. The potential uses of this tool for studying the epidemiology of disease are discussed.

Molecular marker for detection and diagnosis among the isolates of Rhizoctonia solani (AG-4) in Taiwan by RAPD-PCR

L.C. Chen¹, M.G. Chiu¹and C.W. Chen²

¹Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, R.O.C.;²Taichung District Agricultural Improvement Station. Council of Agriculture, Taichung

The isolates of Rhizoctonia solani AG-4 cause damping off disease of vegetables, flowers, and field crops, especially in seedlings in Taiwan. Due to lack of marker in R. solani AG-4 that difficulty for detection R. solani AG-4 population in the field. In this study we have explored the possibility of using DNA polymerase as genetic marker for R. solani AG-4. Collected the isolates around Taiwan and preserved on the PDA plate. By the RAPD analysis of R. solani AG-4 isolates was undertaken to elucidate its genetic complexity, as well as to identify a specific DNA fingerprint for R. solani AG-4. A partial genomic library of isolates MG-01 of R. solani AG-4 was constructed with TOPO-II and transformed into E. coli for the specific primer selection. Using RAPD amplify genomic DNA from isolates of R. solani AG-4 always resulted in a 800bp fragment with OPA14(TCTGTGCTGG) primer. The clone sequencing from the isolate MG-01 of R. solani AG-4.The DNA probe was highly specific among all tested probes. It showed strong hybridization signal with all isolates R. solani AG-4, but no signal on the other fungi or other group isolates tested. A polymerase chain reaction primer pair, derived from a RAPD fragment specific to the isolates of ecological population studies isolated around Taiwan, proved to be suitable for field studies.

Detection of the pathogen causing Eradu patch of lupins

G.C. MacNish and P.A. O’Brien

School of Biological Sciences & Biotechnology, Murdoch University, Perth, 6150, Australia

The field symptoms of Eradu patch, a recently identified disease, are distinct stunted patches in narrow-leaf lupin and ill-thrift patches in barley. The pathogen, a Thin Binucleate Rhizoctonia (TBR), is difficult to isolate. This problem and the ease with which the symptoms can be confused with Rhizoctonia bare patch (R. solani AG-8) have made incidence surveys impractical. To develop an assay specific to TBR, the ribosomal RNA gene region was amplified using ITS primers 1 and 4, and a 610 bp section including the conserved 5.8s region was sequenced. This sequence failed to match any published sequence. Primers specific to TBR were constructed. The specificity of the assay was confirmed using 101 isolates of TBR obtained from many sites in WA. Tests against R solani AG-8 and other fungi isolated from roots were negative. Detection of TBR in roots from artificially infected lupin and barley were positive. However, detection of TBR in root samples collected from field patches in 1997 and 1998 was disappointing low. It was not clear whether this was due to samples being collected at the wrong time or the presence of some type of inhibition. Using new methods for extracting the TRB DNA, including the use of soil immersion plates, gave excellent levels of detection of TBR in 1999.

Detection of R. solani AG2-2 type LP from zoysiagrasses exhibiting symptoms of large-patch using PCR with specific primers

T. Toda, T. Mushika, K. Kageyama and M. Hyakumachi

Faculty of Agriculture, Gifu university, Gifu 501-1193, Japan

Large-patch symptom on warm-season turfgrasses is known as one of the most important turfgrass diseases. The causal pathogen of this symptom has been proposed as R. solani AG2-2 type LP (Hyakumachi et al., 1998). Although many turfgrass diseases are caused by different pathogens, their symptoms are very similar, and a visual diagnosis in the turfgrass fields is not reliable in detecting the causal agent. Thus, a rapid methodology for the exact identification is required. In this study, detection of R. solani AG2-2 type LP from zoysiagrass exhibiting a large-patch symptom was performed using PCR with specific primers. Specific primers, named as A091-F/R, were designed from the sequence specified from the RAPD-PCR product using primer A09. RAPD-PCR using primer A09 obtained only one product from LP isolates and this product was effective in differentiating LP from other two cultural types (ⅢB and Ⅳ) of R. solani AG2-2. At 65℃ annealing temperature and 300mM of dNTP concentration, primers A091-F/R yielded the same product from all DNAs of LP isolates but not from DNAs of ⅢB and Ⅳ isolates. Moreover, these primers also did not yield the product from all DNAs of other R. solani (AG1-10 and BI) isolates and other turfgrass disease pathogens (binucleate Rhizoctonia AG-DI, AG-DⅡ, R. circinata var. circinata, var. oryzae, var. zeae, Pythium graminicola, P. irregulare, P. iwayamai, P. periplocum, P. torulosum, P. ultimum, P. vanterpoolii and Gaeumannomyces graminis var. graminis). PCR product from DNAs of diseased leaves showing large-patch symptoms using primers A091-F/R was in accordance with that of LP isolates. These results suggest that the specific primers A091-F/R are useful for rapid detection of R. solani AG2-2 type LP from warm-season turfgrasses exhibiting large-patch symptoms.

Session 6: Breeding and Induced Resistance

Non-pathogenic Rhizoctonia species elicit systemic induced resistance to Rhizoctonia solani and Alternaria macrospora in cotton

S. Jabaji-Hare¹ and S.M. Neate²

¹Plant Science Department, Macdonald Campus of McGill University, 21,111 Lake shore, Ste.-Anne-de-Bellevue, Canada, H9X 3V9; ²CSIRO, Land and Water, Waite Rd., Urrbrae, South Australia 5064

Recent reports have shown induction of resistance to Rhizoctonia root rot using non-pathogenic strains of Rhizoctonia species (np-R). This study evaluates in greenhouse trials, the biocontrol ability of several np-R isolates against root and foliar diseases of cotton, provides evidence for ISR as a mechanism in the biocontrol and compares in cotton their disease control efficacy with the chemical inducer Bion®. Pretreatment of cotton seedlings with np-R isolates provided good protection against pre- and post emergence damping-off caused by a virulent strain of R. solani (AG 4). Seedling stand of protected cotton seedlings was significantly higher (P < 0.05) than that of non-protected seedlings. Several np-R isolates , significantly reduced disease severity. Bion® treated cotton seedlings did not give protection against seedling rot, but foliar sprays of Bion® combined with pretreatment of cotton with np-R isolates provided good protection. Significantly less leaf spot symptoms caused by Alternaria macrospora occurred on cotyledons pretreated with np-R or sprayed with Bion®, however the np-R treated seedlings had significantly less leaf spot than Bion® treated seedlings. The results demonstrate that np-R isolates can protect cotton from infections caused by root and leaf pathogens through induced resistance and disease control was superior to that observed with a chemical inducer.

Sensitivity of Rhizoctonia solani to derivatives of natural resorcinols of plant origin

R. Zarnowski¹, S.J. Pietr¹ and A. Hendrich²

¹Department of Agricultural Microbiology, Agricultural University of Wrocław, ul. Grunwaldzka 53, 50-357 Wroclaw, Poland; ²Department of Biophysics, Medical University of Wroclaw, ul. Mikulicza-Radeckiego 9, 50-368 Wroclaw, Poland

A group of naturally occurring polyketide-derived phenols, 5-n-alk(en)ylresorcinols (ARs) appear in the Gramineae. Cereal ARs were found to be mixtures of saturated, monoenoic and dienoic homologues with 13-29-carbon chains. Due to their strong antibacterial and antifungal activity, those ARs are biosynthesised especially to protect the plant against phytopathogens and predators. The aim of this study was to determine the in vitro inhibitory effect of orcinol, mixture of saturated ARs from rye and AR with 15 carbon chain on the development of Rhizoctonia cerealis and Rhizoctonia solani. The tested compounds shown significantly different antifungal activity. The ID₅₀ of tested mixture of saturated ARs was in the range from 8.7 to 9.5 mg ml^-1 and was slightly higher than for pure orcinol (ID₅₀ 9.6 – 13.5 mg ml^-1). Recorded values of saturated resorcinol with C 15 chain were significantly higher and varied in the range from 20.3 to 25.8 mg ml^-1). Our date shown higher sensitivity of Rhizoctonia to ARs that other fungi likes Aspergillus, Pencillium and Fusarium. Additionally, we would like to suggest that the mixture of saturated ARs show synergistic effect in comparison with single components. To determine the mode of action of ARs we conducted the study of their influence on biological membranes. The microcalorimetric measurements revealed that high concentration of orcinol (50 mole%) abolish the pretransition of DPPC (dipalmitoylphosphatidylcholine), and alter the parameters of chain-melting transition of this lipid. The decrease of transition temperature, enthalpy, and cooperativity were recorded. From the character of observed changes we conclude that orcinol interacts with DPPC and intercalates the hydrophobic core of lipid bilayer. Those results prove that tested compounds have influence on physicochemical properties of biological membranes of Rhizoctonia.

Molecular approaches for the management of rice sheath blight

R. Velazhahan^{1
& 2}, P. Vidhyasekaran¹, S. Doraisamy¹, S. Muthukrishnan²

¹Department of Plant Pathology, Centre for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India; ²Department of Biochemistry, Kansas State University, Manhattan, Kansas-66506-3702, USA

Rice sheath blight caused by Rhizoctonia solani Kuhn is a serious disease in all rice-growing countries. Resistance gene for sheath blight has not been reported from the existing cultivars of rice so far. Conventional control of this disease mainly depends on the use of chemical fungicides which are causing public concern about food safety and environmental pollution. Recent progress in gene transfer technology makes it possible to produce sheath blight resistant rice varieties by introducing genes which encode proteins with antifungal activity into the rice genome thereby enable the plant to protect itself against fungal attack. With this objective we isolated and characterized a new thaumatin-like protein (TLP) cDNA clone from rice cDNA library prepared from R. solani- infected rice plants. The cDNA contains an open reading frame of 693 bp encoding a 231 amino acid protein with a molecular mass of 24.4 kDa. Using this TLP gene, we developed transgenic rice plants constitutively overexpressing TLP at higher levels. Bioassays of transgenic rice plants challenged with the Rhizoctonia solani, indicated that overexpression of TLP resulted in enhanced resistance compared to control plants. To study the early molecular events underlying the expression of disease resistance in rice, we isolated and partially purified a high molecular weight elicitor molecule from the mycelial walls of R. solani. The elicitor not only induced cell and tissue necrotization in rice but also defense-related activities such as activation of antioxidants and scavengers of active oxygen species. We purified and characterized a 35-kDa chitinase from suspension cultured rice cells. The purified chitinase inhibited mycelial growth of R. solani in vitro and caused marked swelling and lysis of fungal hyphal tip. We isolated and characterized a host-specific toxin from the culture filtrate of R. solani. This toxin suppresses defense mechanisms of rice. The results of these investigations will be discussed.

A study on resistance to Rhizoctonia disease mutant form potatoes, selected by biotechnology on Rhizoctonia culture

E.M. Shaldyatva¹, N.S. Leonova²

¹Novosibirsk Agricultural University, 630117 Ivanova 45–34, Novosibirsk, Russia; ²Institute of Cytology and Genetics, Siberian division of Russian Academy of sciences, Novosibirsk, Russia

Rhizoctonia disease of potato is very severe in Siberia. Yield losses make up 17–30%. Because Rhizoctonia infection is transmitted through tubers and soil, it is very difficult to control the infection by chemical methods. Breeding for resistant potato to Rhizoctonia disease is clearly advantageous. We performed many year experiments in which mutant potato 7 varieties were selected on selective medium with culture filtrate of Rhizoctonia solani AG-3 strains differing in phytotoxicity. The potato plants, that survived on the selective medium with one of the R. solani strains were transferred to the selective medium with the second strain. The plants that survived on the two selective media were planted in the greenhouse where they were estimated (by Frank`s estimate scale) for resistance in artificially Rhizoctonia infected soil: 0—20% versus 60—80% in controls. Rhizoctonia metabolite content in the selective medium, the higher Rhizoctonia resistance of plants: in the selected was. Our method provides rapid development and estimation of improved Rhizoctonia resistant potato.

Transformation of barley for resistance to Rhizoctonia root rot with the codon-optimized chitinase gene from Trichoderma harzianum

Y. Wu¹, R.J. Cook^1,2, H. Horvath², C.G. Kannangara², D. von Wettstein^2,3

¹Dept. of Plant Pathology; ²Dept. of Crop and Soil Sciences; ³Dept. of Genetics and Cell Biology, Washington State University, Pullman WA 99164, USA

Rhizoctonia root rot of barley, caused by Rhizoctonia solani AG8, is the most important root disease of wheat and barley grown with reduced tillage (no-till) in the Pacific Northwest USA. This same pathogen also attacks all broad of crops available to farmers in this region and therefore can not be controlled by crop rotation. The objective of this study is to test the hypotheses that barley genotypes can be developed with resistance to Rhizoctonia root rot by introducing a 41 kDa optimized endochitinase gene from Trichoderma harzianum into the barley genome. The coding region of the gene cThEn42 (1272bp) was accordingly synthesized to contain a G+C content of 65.1% as required for transgene expression in barley. The signal peptide sequence (SP) of HVCHI26 was introduced upstream of cThEn42(GC) for extracellular distribution. Twenty-eight oligonucleotides covering by overlaps both strands of the synthetic gene were assembled into the gene SP+cThEn42 (GC). This gene was inserted together with the bialaphos-resistance gene bar under the control of the ubiquitin promoter and nos terminator between the T-DNA left and right border to yield plasmid pYW100 and transformed into Agrobacterium tumefaciens strain AGL1. Transformation by co-cultivation with immature zygotic embryos yielded 140 transgenic barley plants verified by PCR analysis. Assays for endochitinase activity and resistance to R. solani AG8 are proceeding.

Induction of chitinase among rice cultivars infected with Rhizoctonia solani

C.L. Shrestha¹, I. Oña², S. Muthukrishnan³ and T.W. Mew⁴

¹Division of Plant Pathology, Nepal Agricultural Research Council, P.O. Box 1126, Khumaltar, Lalitpur, Nepal; ²Entomology and Plant Pathology Division, International Rice Research Institute, M.C.P.O Box 3127, Makati City 1271, Philippines; ³Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA; ⁴Entomology and Plant Pathology Division, International Rice Research Institute, M.C.P.O Box 3127, Makati City 1271, Philippines

Shrestha, C. L., Oña, I.P., Muthukrishnan, S and Mew, T.W. Induction of chitinase activities among rice cultivars moderately resistant and susceptible to Rhizoctonia solani infection.

One-hundred-thirteen rice cultivars were selected and screened for sheath blight resistance in the greenhouse. Cluster analysis based on RLH (%) generated 4 groups of cultivars at 3.27 coefficient of similarity. The chitinase activity of the moderately resistant cultivars Betichikon, Dudruchi, Khato Chalani, Padi Pulut Malat, Kakua, IR72, Khakibinni, and Khajur Kandi was induced at 24 hr after inoculation but in susceptible IR58, the chitinase activity was induced at 36 hr after inoculation. Likewise, western blot analysis showed that the 28 and 35 kDa chitinases were induced and increased upon Rhizoctonia solani infection in these cultivars. The percent relative lesion height (% RLH) and the number of infection cushions were negatively correlated with the level of chitinase activity. The moderately resistant rice cultivars had high level of chitinase activity but had lower disease severity and lower number of infection cushions formed.

Relationship between resorcinol content in seeds of barley and seedling infection by Rhizoctonia

S.J. Pietr and R. Zarnowski

Department of Agricultural Microbiology, Agricultural University of Wrocław, ul. Grunwaldzka 53, 50-357 Wroclaw, Poland,

The naturally occurring polyketide-derived phenols, 5-n-alk(en)ylresorcinols (ARs) showed significant antifungal activity in vitro versus Rhizoctonia cerealis and Rhizoctonia solani. Due to their strong antibacterial and antifungal activity, those ARs are biosynthesised to protect the plant against pathogens. The objective of this study was to determine the sensitivity of barley cultivars, which display significantly different concentrations and quality of ARs in the waxy epicuticular layer of grain. The concentration varied from 38.5 to 108.5 mg kg^-1. Barley seeds were sown in sterile sand artificially infested with Rhizoctonia cerealis and Rhizoctonia solani. The infection of barley seedling was monitored during first five days. The seedlings of cultivars with higher amount of ARs in waxy epicuticular layer were infected in smaller degree. The comparison of seedlings of the same cultivar with various quantities of ARs has also proved our observations mentioned above.

The result has confirmed the antifungal activity of analysed phenolic compounds against Rhizoctonia in vitro. The ability for production of saturated resorcinols could be taken into account as one of possible factor during plant breeding.

A method for screening virulent rice sheath blight pathogens and selecting mutant disease-resistant lines of rice

C.H. Chang¹, Y.Z. Lin¹, C.S. Wang² and L.C. Chen¹

C.H. Chang¹, Y.Z. Lin¹, C.S. Wang² C.M. Huang¹ and L.C. Chen¹

¹Dept. of Plant Pathology, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan, ROC; ²Dept. of Agriculture, Taiwan Agricultural Research Institute

Different from the traditional disease-resistant assay waited for a long time, we used the separated leaf sheath to get the results of tests and compared with the relationship between traditional disease-resistant assay and the new method. This method can save much time but get the same results. We cut same-age and same-height leaf sheaths into same-length pieces and put into 18-cm tubes with 0.5g cotton and 8ml water. The sheaths were inoculated at basal regions by sclerotia and under the growing-fluorescent lamps for 28°C 5 days. The control sheaths could maintain green and not roll up. Then we use the chemical mutant lines (more than 700 mutant lines) and proceed the disease-resistant assay with the highest virulent isolate. The whole results must be known after the end of the symposium. An indoor selection and at least two field tests will be proceeded. We believe that there should be some disease-resistant lines and then will request other organizations to determine other characteristics and expect that the disease-resistant cultivar will reduce the use of chemical pesticides as well as improve the incomes of farmers and natural environment where all humans live.

Preliminary study on resistance to sharp eyespot (Rizoctonia cerealis) in wheat germplasm

S. Cai, Y. Cao, X. Fang, and Z. Zhu

Institute of Food Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014

Wheat sharp eyespot (Rizoctonia ceralis) has been epidemic in recent years and resulted in severe yield losses in winter wheat regions in China. This study was conducted to develop screening technology and to explore resistant genetic resources to sharp eyespot for wheat genetics and breeding in 1996-1999. By inoculating soil with cultured wheat grains with mixed cultures of R. cerealis, typical symptom of the disease appeared at the base of columns and percentage of diseased columns in the sensitive check was as high as 100%. Disease indices in the sensitive check are consistent in the nursery while varieties differed significantly in response to sharp eyespot in terms of disease indices and diseased columns. It indicates that the inoculating technology could effectively identify difference of resistance to wheat sharp eyespot in wheat. Several resistant varieties (disease index was smaller than 10%) were screened out. Generally, introduced cultivars showed the strongest resistance, followed by land-races. Improved varieties seem to be the most virulent in this study. It could be contributed to narrow genetic base and most improved varieties have similar parents so that the disease is customized to the genetic constitution in cultivars. In order to control the disease with genetic means, it is essential to wide genetic base and use the resistant germplasm to breed resistant commercial varieties. Genetic sources of resistant materials were analyzed according to their pedigrees.

Evaluation of inoculation methodologies of Rhizoctonia aerial blight of soybean in Brazil

M.C. Meyer¹ and N.L. de Souza²

¹Brazilian Agricultural Research Corporation, National Center for Research on Soybean (Embrapa Soybean), Balsas, MA, Brazil; ²Sao Paulo State University, College of Agronomic Science, Department of Crop Production (UNESP-FCA) Botucatu, SP, Brazil

Four inoculation methodologies of Rhizoctonia solani (AG1-IA) isolated from soybean were tested to evaluate genotype reaction to disease. The treatments were conducted with soybean cultivars ‘Embrapa 63 (Mirador)’ and ‘MA/BRS 165 (Seridó RCH)’, at V5 growth stage, in whole plant (2 plant/pot, 4 repetitions) and in detached trifoliate leaves (3 repetitions) and consisted of: (1) inoculation with 5-mm-diam mycelial disk from 2-days-old culture on PDA, at 27^ºC in darkness (1 disk for leaf); (2) spraying of mycelial suspension, obtained from cultivation on potato-sucrose-peptone liquid media, combined with water at rates of 1:3, 1:6 and 1:30 (v/v), blended and triturated at high speed for 30 s; (3) spraying of powder mycelia formula, obtained from colonized eatable rice, restored in water at rate of 5%; (4) spraying of powder-talcum-mycelia formula (25% concentrated), obtained from fungus culture in liquid media, restored in water at rate of 5%. The volume of inoculum sprayed was 2.5 mL/plant and 1.5 mL/detached leaf. Disease severity scales were used to evaluate whole plant (Harville et al., Soybean Genetics Newsletter 24:188, 1997) and detached leaf (Muyolo et al., Plant Disease 77:234, 1993). The best results were that obtained in treatment 2, with 1:6 mycelial dilution. Mycelial disk inoculation was very drastic, showing high severity rates and it hindered to distinguish possible genetic resistance sources. Treatments 3 and 4 showed very low severity rates (below 5% of infected leaf area) and increased the development of saprophytic fungi on rice fragments. It was not observed differentials reactions between cultivars.

Session 7: Disease Control

Cloning chitinase genes for biological control of Rhizoctonia solani and induced resistance

I. Chet

Otto Warburg Center for Agricultural Biotechnology, The Hebrew University of Jerusalem, Faculty of Agriculture, P.O. Box 12, Rehovot 76100, Israel.

Trichoderma is well-known biocontrol agent of plant pathogenic fungi. Upon contact with the host, Rhizoctonia solani, Trichoderma harzianum coils around the host hyphae. This reaction has been found to be rather specific, and lectin - carbohydrate interactions were assumed to mediate the attachment. As a result of the recognition process, a group of extracellular lytic enzymes is produced, including b-1,3, glucanases, proteases and chitinases. We have introduced the cloned chitinase from Serratia marcescens (chiA) into the Trichoderma genome under the 35S constitutive promoter from CaMV. The transformed Trichoderma secreted high levels of chitinase with no induction by chitin. The transformants showed a remarkably better lysing in Sclerotium rolfsii compared with the wild type. We have used the chiA gene as a probe to isolate a chitinase gene from a cDNA library prepared from T. harzianum (T-35). This gene was cloned in the expression vector Blue script in E. coli. In greenhouse experiments, significant suppression of disease caused by R. solani was detected when bean seedlings were irrigated with the engineered E coli. Similarly, this gene was introduced into Rhizobium meliloti which became antagonistic to pathogenic fungi. ChiA was also introduced into tobacco plant and significantly increased their resistance to R. solani. We have investigated the expression of the various N-acetylglucosaminidases and endochitinases during mycoparasitism of Trichoderma on the plant pathogen R. solani. When Trichoderma was antagonizing R. solani, both the endochitinase and the N-acetylglucosaminidase activities were expressed. We have employed a set of three fluorescent substrates to identify chitinolytic activities of proteins excreted by Trichoderma. The system was found to be composed of six distinct enzymes, one of which has not been reported previously. Application of some microbial genes encoding for active antifungal proteins can serve both for improving biological control as well as to induce resistance in plants.

Etiology and chemical control of Rhizoctonia seedling blight and root rot in chickpea

S.F. Hwang¹, K.F. Chang², B.D. Gossen³, G.D. Turnbull¹ and R.J. Howard²

¹Alberta Research Council, Vegreville, AB T9C 1T4; ²Alberta Agriculture, Food and Rural Development, CDC South, Brooks, AB T1R 1E6; ³Agiculture and Agri-Food Canada, Research Centre, Saskatoon, SK S7N 0X2.

Controlled-environment studies were conducted to examine the etiology of seedling blight and root rot caused by virulent strains of Rhizoctonia solani collected from chickpea fields in Saskatchewan, Canada. A negative log-linear relationship was observed between Rhizoctonia inoculum concentration and emergence, root dry weight and shoot height, while a positive trend occurred between inoculum level and root rot severity. Root rot was more severe at moderate soil temperatures. Nodulation was reduced for severities ³ 1, while ratings above 1 and 3 reduced root dry weight of cvs. Sanford and Tyson, respectively. Seedling emergence and survival in infested soil were generally greater at a seeding depth of 2 cm than 5 cm.

In field trials, inoculum placed with the seed consistently reduced emergence and seed yield relative to the untreated control. Both Desi and Kabuli types were susceptible to seedling blight. Vitaflo 280 + Apron seed treatment improved seedling survival over the noninoculated control. Mechanical damage to seed did not adversely affect seedling survival, but yield was lower in plots planted with damaged seed. Fungicide treatment reduced the impact of seed damage, but did not always restore yield to the same level as undamaged seed.

Towards an integrated control of Rhizoctonia solani in sugar beet

J.H.M. Schneider and W. Heijbroek

Institute of Sugar Beet Research (IRS), P.O. Box 32, 4600 AA Bergen op Zoom, the Netherlands

Rhizoctonia solani seriously threatens sugar beet production in Europe, Chile and the U.S.A. Soil fungicides only have a limited effect on disease reduction. Control through crop rotation seems difficult because of the wide host range of the dominant pathogen, AG 2-2IIIB. A major effect in disease control is to be expected from rhizoctonia resistant sugar beet varieties. Therefore, in 1998 a project was started to test rhizoctonia resistant varieties in various countries in conjunction with a characterization of the anastomosis groups. First results are presented.

In field trials conducted in the Netherlands, fodder radish and white mustard reduced crop loss due to R. solani in a first year trial when applied as a fallow crop. A black fallow also limits disease expression. Young plants of resistant varieties still are susceptible to AG 2-2IIIB and therefore seed pelleting with fungicides or biocontrol agents is necessary for an effective use of resistant varieties. An application of azoxystrobine reduced the number of infected plants and decreased disease severity on average. Resistant varieties varied in resistance when screened in natural infested farmers’ fields.

Biological control of root rot of wheat caused by Rhizoctonia solani

C.S. Pandian and D.V. Singh

Division of Plant Pathology, Indian Agricultural Research Institute, New Delhi 110 012 (India)

Root-rot of wheat caused by Rhizoctonia solani kuhn is more prevalent in areas where rice-wheat cropping system is being followed and late planting is a common practice. No resistant sources are available for this disease and at the same time fungicides control is not eco-friendly and involves high cost. An attempt has been made to develop bio-control strategies using fungal and bacterial agents. In the present study, among the antoagonists tested namely Trichoderma viride, T. harzianum, T. hamatum, Gliocladium virens, Bacillus subtilis, Pseudomonas fluorescens, only two, T. harizanum and P. fluorescens were found to be more effective in suppressing mycelial growth, sclerotial production and germination of R. solani. For testing the efficacy of the bio-agents in vivo they were mass multiplied using talc as carrier and seed treated with different doses individually and in combination. Among the treatments, the combination of T. harzianum and P. fluorescens @ 4 gm each recorded higher seed germination (92.5%) and increased shoot length (25.5 cm) and root length (24.25 cm) which was highly significant over control, after 45 days of sowing. The results showed that the bio-agents were having positive response on the vigour of the wheat plants. The rhizosphere population of T. harizanum and P. fluorescens was higher, in combination @ 4g each than the bio-agents treated individually. The correlation between rhizosphere population of bio-agents and disease incidence was linear and negative as the population of bio-agents increased and the disease incidence decreased.

Biological control of Rhizoctonia-disease (Rhizoctonia solani Kühn) of cropped plants in Romania

T.E. Sesan¹, F. Constantinescu¹, I. Taut²

^[1]Research Institute for Plant Protection Bucharest-Romania; ²Experimental Forestry Station Cluj-Napoca-Romania

Rhizoctonia solani induces root and collar rot in many cropped plants. Trying to find some non-chemical means against Rhizoctonia-disease, 5 Bacillus isolates, 19 Trichoderma viride isolates and mutants and 12 other fungal isolates (Gliocladium roseum, Epicoccum purpurascens, Aspergillus clavatus, Coniothyrium minitans, Ulocladium chartarum, Sordaria fimicola) have been tested against R. solani on bean, vegetables (tomato, cucumber) and conifers. Among Bacillus strains which showed strong antagonistic activity in vitro, 2 isolates - B. subtilis (Bs 1) and B. amyloliquefaciens (Ba 1) -proved the highest efficacy, in greenhouse conditions, conferring the most significant protection of tomato Unirea cv. and cucumber Cornichon cv. seedlings against R. solani.

Among the fungal isolates tested against R. solani, only T. viride proved antagonism against test-fungus. 16 T. viride isolates and mutants tested proved to be highly antagonistic to this pathogen. Application of the fungus T. viride (isolate Td 50) in glasshouse to soil (10-15 g/kg soil) and seed (2-4 g/kg dry biomass) and of 7 mutants originating from the isolate Td 5 (Td C, Td E, Td I, Td K, Td M, Td N) as seed dressing (2-4 g/kg dry biomass) provided protection of bean seedlings Progres cv. from incidence and evolution of disease, similar with the efficacy of chemical standards (Methyl-tiophanate 3 g/kg, AC-8 2 g/kg), in glasshouse conditions (RIPP Bucharest) and in field (Uzlina - Tulcea). Seed dressing of beans with T. viride is recommended to be included as a biological method to develop some Integrated Management Systems.

T. viride, used as a seed treatment of conifers in nursery (Rastolnita - Mures), proved, mainly at a dose of 4 g/kg, efficacy in protecting Pinus nigra, Larix decidua and Picea abies seedlings against damping-off, caused by R. solani, mainly at a dose of 4 g/kg.

Management of banded leaf and sheath blight of maize using Pseudomonas fluorescens

G. Shivkumar and R.C. Sharma

Division of Plant Pathology, Indian Agricultural Research Institute New Delhi-110012, India

Rhizoctonia solani, the organism causing severe blight in several crop like rice, sugarcane and sorghum, hitherto was considered as insignificantly important so far as maize was concerned. However, during the last two decades it seems to have crossed over to maize with a vengeance so much so that it has now become like a scourge in maize growing tracts of South and South East Asia. Banded leaf and sheath blight (R. solani f. sp. sasakii) of maize symptomised by this organisms has now been found to cause losses to the extent of almost 100 per cent, when the ear rot phase of the disease predominates. This alarming situation further aggravated by the lack of and/or failure of identification of resistant genes in the existing germplasm of maize. Thus management of the disease utilizing host resistance has not been successful so far for want of naturally occurring resistance sources in maize.

Managing the disease through chemicals and other cultural means have also not been found to be encouraging. Thus it leaves one with the only other alternative of combating this soil borne fungi through biological means employing fungal-bacterial antagonism as one of the tools. Existence of such antagonism has been reported earlier. In the investigation reported herein, we attempted to use Pseudomonas fluorescens isolate from maize rhizosphere to look into the possibility of not only the inhibition of mycelrial growth of the fungus, but also eventual sclerotial production in vitro. Additionally, we also carried out inoculation of P. fluorescens under field conditions to the maize crop in order to see any advantage to subsequent tolerance to disease spread as well as the growth of the crop and its final productivity. The paper being presented embodies the results obtained on the above aspects.

Application of antagonistic microorganisms in a sandwich design for control of deeding damping off diseases caused by Rhizoctonia solani and Pythium species

T.Y. Chuang¹ and P.J. Ann²

¹Former in Department of Plant Pathology, National Taiwan University, Taipei, 106, Taiwan, ROC.; ²Department of Plant Pathology, Taiwan Agricultural Research Institute, Wufeng, 413, Taichung, Taiwan, ROC

A sandwich method was developed for application of antagonistic microorganisms to control of seedling damping off diseases caused by Rhizoctonia solani. The procedures were designed as follows. Antagonistic microorganisms were added to vermiculate in the concentration about 10^9-10 cfu/ml, and the mixtures were air-dried. A cultural medium was put on the bottom of a pot container. Then one centimeter thick of antagonistic microorganisms-vermiculate mixture was put on the top of the cultural medium as the bottom layer, the middle layer was seed, and the top layer was antagonistic microorganisms-vermiculate mixture again. When isolates of Tp-Tu311 & Tn-y21 of Bacillus subtilis or Y11-2 of Sporobomyces sp., respectively, were applied to cultural media as described above, seedling damping off diseases caused by R. solani significantly decreased. The tested crops included rice, corn, adzuki bean, 15 species of vegetables and 3 flowering plants. The activity of inhibition ability of the three kinds of airy-dried antagonistic microorganisms-vermiculate mixtures could maintain at least for 12 months at room temperatures. Among the three tested isolates of antagonistic microorganisms, Tp-Tu311 isolate of B. subtilis was most effective. As long as the concentration of the antagonistic bacterium in the vermiculate mixture was higher than 10⁶ cfu/ml, the Tp-Tu311 isolate applied as sandwich design was effective in inhibition of Rhizoctonia damping off diseases. Using the sandwich design, the three antagonistic microorganisms were also effective significantly in control of cucumber damping off caused by Pythium splendens, but failed to decrease tomato seedling blight caused by Phytophthora capsici.

Cloning and expression of chitinase gene from Bacillus subtilis CHU26 in Saccharomyces cerevisiae and the biocontrol activity analysis

C.Y Yang and J.S.M. Tschen

Department of Botany, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C.

Chitin, a polymer of N-acetylglucosamine (GlcNAc), is an important element in insect exo-skeletons, crustacean shells, and fungal cell walls. Apart from cellulose, it constitutes the second most abundant polysaccharide in nature. Chitinase (E. C. 3. 2. 1. 14) can degrade chitin into chitosan, not only found in fungi, which contains chitin in the cell walls, but also in the other organisms, including bacteria, yeasts, and plants (most do not contain chitin). It has been proven that the expression of chitinase genes could enhance resistance to pathogenic fungi by degradation of the pathogenic cell wall. Bacillus subtilis CHU26 secretes chitinase into the culture medium, with a stronger extracellular chitinase activity than Bacillus circulans WL-12. A 4kb DNA fragment of B. subtilis CHU26 was cloned from the genomic DNA library using Southern hybridization with B. circulans WL-12 chiA1 as a probe. After continuous screening and subcloning of the library, a complete open reading frame, which had 1791 nucleotides and a protein encoded with 596 amino acids was determined. The deduced amino acid sequence had alignments to the other chitinases, which all had the same catalytic domain. The B. subtilis CHU26 chi could be expressed in Escherichia coli to form a clear zone on the colloidal chitin plates with a stronger chitinase activity than B. circulans WL-12. When the chi was subcloned into the shuttle vector pAAH5, it could also be expressed in the Saccharomyces cerevisiae AH22. In an infection test using radish seedlings and Rhizoctonia solani CH341, the B. subtilis CHU26 chi could inhibit the pathogenic activity of R. solani CHU341.

Production and formulation of Trichoderma spp. biomass used to control leaf blight of Bambarra goundnut (Vigna subterranea) caused by Rhizoctonia solani in the South of Thailand

A. Pengnoo, V. Somsap, A. Chumthong, P. Sawangsri, A. Nooduang and M. Kanjanamaneesathian

Faculty of Natural Resources, Prince of Songkla University, Hat Yai Campus, Songkhla 90110, Thailand

This project aims to formulate and produce an effective Trichoderma spp. biomass technology to control leaf blight disease in Bambarra groundnut caused by Rhizoctonia solani. This disease is a major disease in Southern Thailand because environmental conditions are suitable for rapid disease development. Production inputs such as fertilizer and fungicide applications for leaf blight control are minimal because this crop are cultivated as intercrop with other main crops poor soils by low income farmers. The Trichoderma spp. biomass has been produced with applicable technology which will be consequently transfer to farmers who can produce and formulate Trichoderma spp. biomass with basic requirement by their own. Trichoderma spp. biomass was initially produced by transferring the fungus to flask containing sterile pericarp of oil palm mixed with urea fertilizer (10:1, w/w). The fungus was incubated at room temperature at 26-30 ⁰C for 7 days and then mixed with dolomite (pH 8.2 , 325 mesh) (1:1, v/v) in Waring blender. The formulation was stored in plastic bag and kept in an air tight plastic container and stored in the refrigerator at 6 ⁰C. To assess the shelf life of the end product, colony forming unit (cfu) per gram of the formulation was carried out every 14 days. After 3 months of storage in the refrigerator, the formulation contained 1 x 10 ¹⁰cfu per gram. The further testing to determine longer shelf life has been studied. Research is in propers to test the developed Trichoderma spp in biomass and to determine its efficacy in controling leaf blight disease in pot and field experiments.

Biological control properties of a new Rhizoctonia-like species (BNR), Ceratobasidium albasitensis isolated in Spain

V. González, M.A. Portal, J. Acero, J. Sánchez-Ballesteros and V. Rubio

Centro Nacional de Biotecnología (CSIC-UAM). Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.

Rhizoctonia diseases of vegetable, ornementals, trees, nursery and greenhouse crops cause large economic losses to the agricultural industry. Many isolates of R. solani Kühn and other taxa from the so-called Rhizoctonia complex attack a wide range of hosts, producing several pathologies such as seeds decay, damping-off of seedlings, stem cankers, root rots, fruits decay, etc.

Some non-pathogenic or hypovirulent isolates within the form-genus, usually denominated Binucleate Rhizoctonia (BNR), have been shown to be highly effective biocontrol agents. In most of studies up to date, all the known isolates capable to prevent and protect several plant diseases have been found to belong to genus Ceratobasidium Rogers (anamorph: Ceratorhiza R.T. Moore). Moreover, several studies have shown that some of the non-pathogenic BNR isolates were also promoted plant growth, even in the absence of the pathogen. Several authors had pointed that protective isolates usually grow relatively slower than virulent ones, what could represent a disadvantage for their use as biocontrol agents. However, it is suggested to apply them as close as possible to the seeds and very early in the process.

The present contribution shows results from protection tests performed with several isolates belonging to a new BNR species: Ceratobasidium albasitensis V. González & V. Rubio isolated in Spain. The tests were against several plant pathogens including R. solani AG 4 isolates and common pathogens such as Alternaria, Fusarium, Penicillium, Acremonium, etc. Protection tests were carried out on a wide range of plant species, including wheat, alfalfa, rape, carrot, tomato, grass, onion, garlic and pine.

Prior to the protection tests, both the supposedly protective isolate and the pathogens selected were assayed for pathogenicity against the different plant species tested. All the experiments were performed in petri dishes in the laboratory, except for pine and wheat that were carried out at laboratory and greenhouse scale.

Results showed that protection effects generally depend on the plant species assayed, as well as on the pathogenic fungi selected for each experiment. As expected, the timing for inoculation of the pathogens with respect to the protective strains was also an important parameter for the improvement of the protection effects. Thus, consecutive inoculation of the pathogen 2-3 days after the BNR strains allowed colonization of the root and stem surfaces by the protective fungi, preventing the attack of the pathogen probably by differential competition for infection sites. Finally, growth promotion was tested only in garlic assays, and effects were determined by visual comparisons with controls and by estimation of biomass (aerial and radical) in terms of dry weight. A considerable increase of biomass was observed in general but more significant is the increase in radicular mass of the plants.

Efficacy of some fungicides in controlling root rot of French bean caused by Rhizoctonia solani Kuhn

M. Mozaffar Hossain, M.H Rashid and D.P. Singh

Horticulture Research Centre, Bangladesh Agricultural Research Institute, Joydebpyr, Gazipur-1701, Bangladesh

Root rot caused by Rhizoctonia solani Kuhn is one of the major diseases of French bean (Phaseolus vulgaris L.) in Bangladesh. The disease occurs at the seedling stage attacking either at the root system or the foot of stem at the collar region. The infected plant dies within a few days. The study involving two experiments was undertaken to find out the efficacy of some fungicides in controlling the root rot of French bean. It was conducted at the central research station of Bangladesh Agricultural Research Institute, Gazipur, Bangladesh during 1999-2000. There were five fungicides, such as, Vitavax-200 (Carboxin), Bavistin (Carbendazim), Dithane M-45 (Mancozeb), Rovral 50WP (Iprodione), and Tilt 250EC (Propiconazole) used for treating the seeds in the 1st experiment. In another experiment, seeds were treated as well as soil drenched by Bavistin and Vitavax-200. It was noticed that seed treatment along with soil drenching was much more effective than the seed treatment alone. However, the lowest incidence (13.67%) of root rot was recorded when seeds were treated by Tilt 250EC followed by Bavistin (13.70%), Rovral 50WP (14.33%), Vitavax-200 (17.69%), Dithane M-45 (21.67%) and untreated control (29.55%) in the 1st experiment. In the 2nd experiment, the least incidence of root rot was noticed when seeds were treated and soil drenched with Bavistin (5.23%) followed by Vitavax-200 (11.87%) compared to the control (27.89%). Bavistin @ 0.1% may be recommended for seed treatment and soil drenching in controlling root rot of French bean.

Situation and Control of Vegetable Diseases Caused by Rhizoctonia spp. in Hubei Province

C.H. Zhou

Department of Plant Protection, Huazhong Agricultural University, Wuhan, Hubei 430070,P.R.China

In Hubei, serious vegetable diseases were caused by Rhizoctonia spp. More than 20 varieties vegetables could be infected by Rhizoctonia spp. There are three characters in these diseases: wide-ranging distribution, huge loss in products and quantity, difficult to control. The symptoms include seedling rot, stembase rot, and fruit rot. Two Rhizoctonia species could infect vegetables, R. cerealis and R. solani. Vegetable species showed different resistance to Rhizoctonia. These diseases were related with species resistance, cultural system, temperature and relative humidity .The control methods include seedlingbed sterilization, good cultivation, and chemical control. Resistance breeding is the best way to control these diseases. We want to clone some R-genes to Rhizoctonia spp. from Arabidopsis.

Occurrence and control of rice sheath blight in Korea

C.K. Kim

Plant Pathology Division, Crop Protection Department, National Institute of Agricultural Science & Technology, Rural Development Administration, Suweon, Korea

Rice sheath blight caused by Rhizoctonia solani is the most serious rice disease in terms of affected planting area and yield reduction. Once the infection starts, the disease spreads fast, horizontally as well as vertically, within a short period and it continues until maturity. However, the symptom is quite often overlooked and farmers are apt to lose proper timing to control. Rice cultivars with short stature tend to have more damage than tall cultivars. Degree of damage at maturity varied depending upon weather conditions. It averaged 30.5% in the standard nitrogen plot and 40.5% in the double nitrogen plot from 1976 to 1995 crop seasons. Yield loss was greater when the lesions reached upper parts of rice plants. It can reach 50% when the lesions develop on the flag leaves and/or sheaths. Control of the disease can be done through removal of over-wintered sclerotia before transplanting and chemical spray at time when percent diseased hills reaches 20%.

Study on the biological control of the mycorrhizal fungi to damping off (Rhizoctonia solani) of pine seedlings

M. Tang

Northwest Sci-Tech University of Agriculture & Forestry, Yanling, Shaanxi, 712100

The seedlings of Pinus tabulaeformis were inoculated with micorrhizal fungi (Suillus grevillei), the result shows that mycorrhizal fungi may not only promote the growth of pine seedlings, but also protect them from infection of damping off disease, the mortality of pine seedling damping off disease were decreased by 15.6-22.1%. Mycorrhizal fungi were found to be able to destroy the vegetative mycelia of R. solani, and inhibited the formation and germination of conidia, sporangia and sclerotia of pathogens.

Sensitivity of Rhizoctonia solani towards sheep urine

J. Raja and V. Kurucheve

Department of Plant Pathology, Faculty of Agriculture, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India

Background and Objectives: Indiscriminate use of synthetic chemicals has caused potential threat to human health, increase in pathogen resistance / mutation and are often costly and not available to common farmers in developing countries. Sheep penning is a common practice followed in rural farming. Animal excrements are known possess pesticidal value. This has prompted us to investigate the toxicity of sheep excrement, urine, against Rhizoctonia solani Kuhn, causing sheath blight of rice under in vitro.

Materials and Methods: Freshly collected sheep urine was used as such forming the standard solution. The antifungal effect was evaluated against R. solani by poisoned food technique.

Results: Sheep urine (10%) recorded total inhibition of R. solani mycelial growth, production and germination of sclerotia in both solid and liquid media and it was at par with fungicide, propiconazole (0.1%). Even after 30 days of incubation, the inhibited mycelial discs and sclerotia did not germinate again when placed on fresh media. Thus, the fungicidal nature of sheep urine was documented. The toxicity was not affected by various physical factors viz., heavy inoculum, autoclaving, temperature (90ºC) and storage (>2 years) under refrigerated condition. There was no change in pH of the sheep urine amended broth. Hydrolytic enzyme production of the pathogen was also greatly reduced. Seed treatment with sheep urine enhanced the seed

germination and vigour of paddy seedlings.

Rhizoctonia-suppressive composts and potting soil

P.H.J.F. van den Boogert, M. Montanari, and J. Postma

Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands

Compost is used in agri- and horticulture as fertiliser or to improve the physical structure and to enhance the disease suppressiveness of the soil. The level and reproducibility of disease suppressive properties of compost and other organic substrates, like potting soil, might be increased by strategic introduction of antagonists during processing and production. In the present study, the establishment and suppressive activity of the antagonistic fungus Verticillium biguttatum was evaluated towards Rhizoctonia diseases in various compost and potting products.

The population dynamics of the antagonist was examined during 3 months of storage at room temperature upon amendment at 10⁵ spores per g product. The suppressive potential of the compost and potting soil was assessed in sugar beet seedling test with R. solani AG 2-2IIIB and in potato tuber test with R. solani AG 3.

Verticillium biguttatum survived at relatively high levels in green waste compost and potting soil but the population faded out in the household waste compost. In the sugar beet and potato bioassays, the disease suppressive activity of the green waste compost and potting soil was improved by antagonist amendment, resulting in less hypocotyl infection and less black scurf. Contrary, household composts were similarly suppressive to both R. solani and V. biguttatum, rendering no improvement disease suppression. Green waste compost and potting soil are apparently non-hostile substrates for antagonist establishment and disease expression. We therefore see great potential for upgrading composts and other organic substrates for disease control by strategic amendment of antagonists or other beneficial micro-organisms.

Studies on bioactivities and antibiotics produced by Paecilomyces marquandii CF110

C.J. Chen¹ and J.S.M. Tschen²

¹Food Industry Research & Development Institute, Hsinchu, Taiwan; ²Department of Botany, National Chung-Hsing University, Taichung, Taiwan

Antagonistic fungi Paecilomyces marquandii. CF110 isolated from Fusarium wilt suppressive soil was studied and its antibiosis effects on Rhizoctonia solani are discussed. Paecilomyces marquandii produced antibacterial and antifungal antibiotic metabolites when grown in soybean meal broth at 32℃ and pH 6.0 for five days. The antibiotic metabolites could be extracted with ethyl acetate. The best solvent system for thin layer chromatography was toluence/ethyl acetate/formic acid (5:4:1, v/v). After elution two antibiotic metabolites Rf 0.35 (A) and Rf 0.42 (B) could be obtained. The antibiotics were purified by column chromatography. Substance A was a yellow powder (dec. mp 185℃). Substance B was a reddish brown powder (dec. mp 287℃). According to the UV, IR, NMR spectrums and the reagent color reaction on TLC-chromatography, the antibiotics were not similar to those produced previously by other fungi. These antibiotics have a broad spectrum and are able to eliminate bacteria and many fungi. The culture filtrate and crude antibiotics had a toxic reaction to tomato and lettuce. The crude extractions could be used experimentally for the bio-control of damping-off in rice seedlings caused by Rhizoctonia solani. Moreover, when rice kernels were coated with Paecilomyces marquandii spores CF110 with methylcellulose, the rice could be protected from Rhizoctonia solani infection (No.3 & TY).

Expression of chitinase A1 gene from Bacillus circulans WL-12 and the application of biological control on Rhizoctonia solani

K.M Hsu, Y.J. Chen and J.S.M. Tschen

Department of Botany, National Chung Hsing University, 250 Kuokuang Road, Taichung, Taiwan 40227, R.O.C.

The chitinase A1 showed a strong affinity to chitin and was suggested as playing a major role in the degradation of chitin in the chitinase system of Bacillus circulans WL-12. We used PCR to amplify the chitinase A1 gene from the genomic DNA of B. circulans WL-12. The amplified gene was then subcloned into E. coli JM109 and S. cerevisiae AH22. Chitinase activity was measured using fluorometric assay using 4-Mu-(GlcNAc)₃ as a substrate. We found a significant amount of chitinase accumulated in the periplasmic space of the E. coli transformant EYCA1. During the late log phase, chitinase activity was measured at about 500 nmoles 4-Mu/h ml in the periplasmic space of the E. coli. The chitinase was secreted into the media after stationary phase of the growth. The chitinase activity was then measured at about 400 nmoles 4-Mu/h ml. The majority of the secretions produced by the chitinase in the S. cerevisiae transformant SACA1 were secreted into the media. The chitinase secretion in the culture media exactly paralleled the growth curve. In this report we describe the construction of a system for the production of chitinase in E. coli and S. cerevisiae. We demonstrate that chitinase could protect seedlings against the plant pathogen Rhizoctonia solani. Moreover, E. coli and S. cerevisiae harbor a plasmid expressed in the chitinase gene of B. circulans WL-12 that could act as a model biocontrol system.

Biocontrol of rice sheath blight and vegetable seedling damping off caused by Rhizoctonia solani by chlamydospore formulation of Gliocladium viride isolate G-8.

R.Z. Huang and S.P.Y. Hsieh

Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan, R. O. C.

A method to produce chlamydospores of Gliocladium viride isolate G-8 under liquid cultural condition was accomplished. The product contained 100% of chlamydospores at concentration more than 10⁸/ml and no any conidia. Field trials conducted in 1999-2000 for 3 consecutive seasons to control rice sheath blight caused by Rhizoctonia solani (AG-1). The results showed that the most effective way was to spray liquid formulation containing chlamydospores at concentration of 10⁵/ml at tillering maximum stage for 3 times. The disease severity decreased more than 80% and yield increased approximately 32%. Application of floatable granules containing 10⁵/g chlamydospores at tillering maximum stage was also very effective to decrease disease severity (more than 60%). Chlamydospores in floatable granules would germinate soon after attached to rice leaf sheath and colonizing there for a few weeks to prevent the infection of the pathogen. Use of mulching paper coating with chlamydospores could both prevent growth of weeds and decrease the severity of sheath blight. By mixing chlamydospore suspension in substrate infested with R. solani (AG-4) could control seeding damping off of radish. However if concentrations of chlamydospores were too high (more than 10⁷/g substrate) would cause inhibition of germination.

Session 1: Taxonomy and Identification

V. Rubio1, V. González1, M. de los Angeles Portal1, J. Acero1, O. Salazar1, M. del Carmen Julián1, M. Hyakumachi2 and B. Sneh3

S.P.Y. Hsieh and R.Z. Huang

P.T. Huang and J.S.M. Tschen

R.C. Fenille1,2, E.E. Kuramae1, N.L. Souza1

G.R. Balali, A.M. Kasalkheh and M. Kowsari

D. González1, M.A. Cubeta2 and R. Vilgalys3

S. Kuninaga 1 and D.E. Carling 2

L. Casadei María1, L. Gasoni1 and M. Rivera2

M.A. Mazzanti de Castañón1, S.A. Gutiérrez1 and L.A. Gasoni2

E.E. Kuramae1,2, A.A.B. Sussel1, R.C. Fenille1, N.L. de Souza1

G. Virgen-Calleros1, V. Olalde-Portugal2, S. Gomez-Sumuano2, R. Hernandez- Matehuala2 and D. Carling3

T.F. Hsieh1, Y.C. Chang1, and C.C. Tu2

G. Tuncer1 and G. Erdiller2

E.C.A. Abeln, D. van Gosliga, O. de Vries, J. Steenbergen, P.H.J.F. van den Boogert, P. Bonants, IJ. Vlug and J.A. Stalpers

E.E. Kuramae1,2, A.L. Buzeto1 and N.L. de Souza1

P.C. Ceresini1,2, H.D. Shew1, R.Vilgalys3, U.L. Rosewich4 and M.A. Cubeta5

M.A. Cubeta1, P.C. Ceresini2,3, H.D. Shew2 and R.Vilgalys4

T. Hashiba, A. Sasaki, K. Katsura, and A. Nagasaka

A.J. Hilton, S.L. Linton and A.K. Lees

B.J. Stodart1,2, S.M. Neate3, E.S. Scott1, K. Ophel-Keller2, P.R. Harvey3 and D.L. Melanson4

G.C. MacNish and P.A. O’Brien

C.W. Chen1, M.G. Chiu2 R.W. Lin2 and L.C. Chen2

C.H. Chang, Y.Z. Lin, Y.M. Chang, F.L. Liu and L.C. Chen

G. Godoy-Lutz, J. R. Steadman, K. Powers and B. Higgings

V.L. Wang and J.S.M. Tschen

R.C. Fenille1,2, E. E. Kuramae1, D. N. Nozaki1 and N.L. Souza1

E.E. Kuramae1,2, D.N. Nozaki1, R.C. Fenille1, N.L. de Souza1

A. Nagasaka, A. Sasaki, K. Katsura, T. Sasaki, and T. Hashiba

Y.H. Tseng, H.C.F. Chow, M.J. Cheu, and J.S.M. Tschen

I.C. Ho, K.Y. Yü, J.N. Lin and J.S.M. Tschen

J.T. Liao, Y.W. Chung J.F. Huang and L.C. Chen

N. Mayek-Pérez2, C. López-Castañeda3, R. Garcia-Espinossa3, M.M. González- Chavira4, J.A. Acosta-Gallegos4, O. Martínez de la Vega5 and J.W. Simpson5

P.C. Ceresini1,2, H.D. Shew1, R.Vilgalys3 and M.A. Cubeta4

S.W. Tung and J.S.M. Tschen

S. Narmatha Lekshmi, J.Y. Wong, T.K. Tan and C.S. Loh

S. Narmatha Lekshmi, T.K. Tan and C.S. Loh

B. Sneh1, and V. Rubio2

C.L. Wang1, J.N. Chu2, C.Y. Chen1 and Y. C. Chao3

L. Manoch1, P. Athipunyakom2 and M. Tanticharoen3

N. Castilla1 and S. Savary2

N. Castilla1 and S. Savary2

Y.S. Lin, K.S. Huang and C.H. Hwang

C.H. Chang, Y.Z. Lin, Z.R. Chang and L.C. Chen

C. Abdul Rauf1, M. Ashraf2 and I. Ahmad3

Factors affecting formation of hymenia and basidiospores of Thanatephorus cucumeris ( teleomorph of Rhizoctonia solani AG-2-2 ⅢB ), the causal agent of Chinese amaranth leaf spot

J.W. Huang1, H.F. Lin1 and T.F. Hsieh2

A. Kamio and K. Inagaki

R. Cardona1, H. Rodriguez1, L. Cedeño2 and H. Nass3.

J.G. Tsay and R.S. Chen

M.A. Cubeta1 and S.M. Neate2

R.R. Mallikarjunaiah and M. Vasanthakrishna

D.L. Melanson1, I. Dumitrescu2 and S.M. Neate2

P.H.J.F. van den Boogert, F.D. Soglio, and A. Luttikholt.

A.K. Lees, D.W. Cullen and A.J. Hilton

L.C. Chen1, M.G. Chiu1and C.W. Chen2

G.C. MacNish and P.A. O’Brien

T. Toda, T. Mushika, K. Kageyama and M. Hyakumachi

S. Jabaji-Hare1 and S.M. Neate2

R. Zarnowski1, S.J. Pietr1 and A. Hendrich2

R. Velazhahan1 & 2, P. Vidhyasekaran1, S. Doraisamy1, S. Muthukrishnan2

E.M. Shaldyatva1, N.S. Leonova2

Y. Wu1, R.J. Cook1,2, H. Horvath2, C.G. Kannangara2, D. von Wettstein2,3

C.L. Shrestha1, I. Oña2, S. Muthukrishnan3 and T.W. Mew4

S.J. Pietr and R. Zarnowski

V. Rubio¹, V. González¹, M. de los Angeles Portal¹, J. Acero¹, O. Salazar¹, M. del Carmen Julián¹, M. Hyakumachi² and B. Sneh³

R.C. Fenille^1,2, E.E. Kuramae¹, N.L. Souza¹

D. González¹, M.A. Cubeta² and R. Vilgalys³

S. Kuninaga ¹ and D.E. Carling ²

L. Casadei María¹, L. Gasoni¹ and M. Rivera²

M.A. Mazzanti de Castañón¹, S.A. Gutiérrez¹ and L.A. Gasoni²

E.E. Kuramae^1,2, A.A.B. Sussel¹, R.C. Fenille¹, N.L. de Souza¹

G. Virgen-Calleros¹, V. Olalde-Portugal², S. Gomez-Sumuano², R. Hernandez- Matehuala² and D. Carling³

T.F. Hsieh¹, Y.C. Chang¹, and C.C. Tu²

G. Tuncer¹ and G. Erdiller²

E.E. Kuramae^1,2, A.L. Buzeto¹ and N.L. de Souza¹

P.C. Ceresini^1,2, H.D. Shew¹, R.Vilgalys³, U.L. Rosewich⁴ and M.A. Cubeta⁵

M.A. Cubeta¹, P.C. Ceresini^2,3, H.D. Shew² and R.Vilgalys⁴

B.J. Stodart^1,2, S.M. Neate³, E.S. Scott¹, K. Ophel-Keller², P.R. Harvey³ and D.L. Melanson⁴

C.W. Chen¹, M.G. Chiu² R.W. Lin² and L.C. Chen²

R.C. Fenille^1,2, E. E. Kuramae¹, D. N. Nozaki¹and N.L. Souza¹

E.E. Kuramae^1,2, D.N. Nozaki¹, R.C. Fenille¹, N.L. de Souza¹

N. Mayek-Pérez², C. López-Castañeda³, R. Garcia-Espinossa³, M.M. González- Chavira⁴, J.A. Acosta-Gallegos⁴, O. Martínez de la Vega⁵ and J.W. Simpson⁵

P.C. Ceresini^1,2, H.D. Shew¹, R.Vilgalys³ and M.A. Cubeta⁴

B. Sneh¹, and V. Rubio²

C.L. Wang¹, J.N. Chu², C.Y. Chen¹ and Y. C. Chao³

L. Manoch¹, P. Athipunyakom² and M. Tanticharoen³

N. Castilla¹ and S. Savary²

N. Castilla¹ and S. Savary²

C. Abdul Rauf¹, M. Ashraf² and I. Ahmad³

J.W. Huang¹, H.F. Lin¹ and T.F. Hsieh²

R. Cardona¹, H. Rodriguez¹, L. Cedeño² and H. Nass³.

M.A. Cubeta¹ and S.M. Neate²

D.L. Melanson¹, I. Dumitrescu² and S.M. Neate²

L.C. Chen¹, M.G. Chiu¹and C.W. Chen²

S. Jabaji-Hare¹ and S.M. Neate²

R. Zarnowski¹, S.J. Pietr¹ and A. Hendrich²

R. Velazhahan^{1
& 2}, P. Vidhyasekaran¹, S. Doraisamy¹, S. Muthukrishnan²

E.M. Shaldyatva¹, N.S. Leonova²

Y. Wu¹, R.J. Cook^1,2, H. Horvath², C.G. Kannangara², D. von Wettstein^2,3

C.L. Shrestha¹, I. Oña², S. Muthukrishnan³ and T.W. Mew⁴