Chakraborty, S.

Charchar, Maria José

Fernandes, C.D.

Guodao, L

Kelemu, Segenet

Ramesh, CR

Weeds, P

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ID # 05-02GENETIC DIVERSITY IN THE ANTHRACNOSE PATHOGEN INFECTINGSTYLOSANTHES IN BRAZIL, INDIA AND CHINAP. Weeds1, S. Chakraborty1, C.D. Fernandes2, M.J. Charchar3, C.R. Ramesh4 L. Guodao5and S. Kelemu61CSIRO Tropical Agriculture, CRC Tropical Plant Pathology, University of Queensland4072, Australia 2 EMBRAPA-CNPGC/Ph.D student UNESP-FCA, Botucatu-SP, 18609-490Brasil 3EMBRAPA-CPAC, Brasilia 73.3000, Brazil 4IGFRI, Dharwad 580005, India5CATAS, Danzhou 571737, Hainan, P. R. China 6CIAT, Apartado Aéreo 6713, Cali,ColombiaAbstractThis work aimed to determine the genetic diversity of Colletotrichum gloeosporioidesinfecting Stylosanthes spp. in Brazil, China and India. A total of 132 isolate originating fromS. seabrana, S. macrocephala, S. capitata, S. scabra, and S. guianensis were used. Four majorgenetic groups were identified from an analysis of genetic diversity using selection-neutralDNA markers. Group 1 contained 20 isolates and this may represent a genotype that migratedfrom the center of diversity in Brazil and Colombia to Australia, Thailand and India. Group 2consisted of 66 Brazilian isolates and group 3 had 19 isolates from Australia, Burundi, Brazil,China, Colombia, Ivory Coast and Peru. The 27 isolates in group 4 were very diverse with>50% dissimilarity between some isolates. Genetic diversity in Brazil and China was moreextensive than in the Indian pathogen population.Keywords: genetic diversity, RAPD, anthracnose, Stylosanthes, international collaborationIntroductionDespite their agronomic suitability, commercial utilization of Stylosanthes spp. hasbeen severely curtailed by anthracnose caused by Colletotrichum gloeosporioides. Thegenetic plasticity of C. gloeosporioides at its center of diversity in South and Central America(Chakraborty et al., 1997; Kelemu et al., 1997) has caused the demise of at least five of theeight cultivars released (Miles and Lascano, 1997). There is a similar history of cultivardevastation by anthracnose in Australia, China, Thailand, The Philippines and other parts ofSouth East Asia (Guodao et al., 1997).Durability of anthracnose resistance may be predicted by the propensity of a pathogenpopulation to change and an improved knowledge of pathogen diversity is essential for this.Selection-neutral Random Amplified Polymorphic DNA (RAPD) markers have been used todemonstrate genetic variation in C. gloeosporioides population infecting S. scabra inAustralia (Chakraborty et al., 1999) and S. guianensis in Colombia (Kelemu et al., 1999).Scant information is available on the genetic diversity of isolates infecting other Stylosanthesspp. in the center of diversity or in any of the countries where these species are increasinglyused for animal production. Through an international collaboration between Australia, Brazil,China, Colombia and India, this work examines the genetic diversity in 132 isolates of C.gloeosporioides originating from S. seabrana, S. macrocephala, S. capitata, S. scabra, and S.guianensis from these and other countries.Material and MethodsIsolates were imported under quarantine permit to the CRC for Tropical Plant Pathologylaboratories at the University of Queensland, Australia, for genetic analysis. Single-spore cultureswere multiplied on ¼ strength clarified V8 juice broth and mycelia and conidia were ground in anextraction buffer (250 mM Tris HCl, 200 mM NaCl, 25 mM EDTA and 5% SDS), centrifuged,cleaned with phenol chloroform, followed by a chloroform wash and DNA was precipitated inethanol. For RAPD analysis DNA from duplicate samples of each isolate was amplified bypolymerase chain reaction for 8 arbitrary decanucleotide primers (Operon Technologies Inc.Alameda, CA94501) in a programmable thermocycler (MJ Research, USA). Amplificationproduct (15 µl) was electrophoresed in 1.6% agarose gel containing 0.024% ethidium bromide.Similarity values were calculated from pooled data using the formula F = 2mxy/[mx +my], where mxy = the number of shared bands and mx and my are the number of bands in eachisolate. The Unweighted Pair Group Method with Arithmetic Mean was used for a clusteranalysis of the similarity values and the SAS procedure ‘TREE’ was used to generate adendrogram (SAS Institute, Cary, NC).Results and DiscussionA total of 92 reproducible RAPD bands ranging from 380 to 2800 bp were obtainedusing the 8 primers. Using a dissimilarity value greater than 25% from the cluster analysis,the majority (105) of the isolates could be grouped into three main groups (Fig. 1). Theremaining group of 27 isolates from Brazil, China, Colombia, Nigeria and The Philippines arevery diverse and many of these are genetically distinct with more than 50% dissimilaritybetween isolates. Majority of these was isolated from S. guianensis, with a smaller numberfrom S. hamata and S. capitata.Group 1 contains 20 isolates from S. capitata, S. fruticosa, S. scabra and S. seabrana.Of these, 3 isolates were from Australia representing the 3 dominant races of Biotype A, 3were from Colombia, one each from Brazil and Thailand and the remaining 12 were fromIndia. This group may represent the original genotype that migrated from the center ofdiversity in Brazil and Colombia to become established in Australia, Thailand and India.Group 2 consists of 66 isolates; all from Brazil and 44 of these are of the samegenotype. Over 80% of group 2 isolates originate from the same set of selected accessions ofS. capitata, S. macrocephala and S. scabra. Similar association between S. guianensis and S.scabra and C. gloeosporioides genotypes has been recorded earlier in Australia (Chakrabortyet al., 1999) and Colombia (Kelemu et al., 1999).The 19 isolates in group 3 come from Australia, Burundi, Brazil, China, Colombia,Ivory Coast and Peru. Eleven of these are from S. guianensis including an isolate of BiotypeB from Australia. It is not known if isolates in this group have similar symptomatology, hostrange and karyotype to the Australian Biotype B. We know, however, that many group 2isolates produce discrete lesions similar to the Australian Biotype A on all host speciesincluding S. guianensis (Perrott and Fernandes, unpublished information). Furtherinvestigation is needed to determine the existence of distinct pathogen Biotypes in Brazil.Due to the small number of isolates studied in this work, it is premature to commenton the genetic diversity of C. gloeosporioides population in China and India. However, somepractical observations can be made. The genetic structure of the pathogen population in Indiaappears to be relatively simple, with less than 10% overall dissimilarity. This may indicatethat the pathogen has established in India following a single introduction. S. fruticosa isbelieved to be indigenous to India and the pathogen population on this host may be morediverse. We have used only 2 isolates from this host and a more extensive collection iswarranted. Nevertheless, a lack of extensive genetic diversity does not imply a limitedpathogenic variation in India. Evidence from Australia suggests that different pathogen racescan arise from the same genetic background (Chakraborty et al., 1999). By comparison, thegenetic structure of C. gloeosporioides population in China is far more complex. The highlevel of dissimilarity between some isolates indicates that the pathogen may have beenintroduced on more than one occasion. Given the relatively short history of Stylosanthesutilization in China, it is unlikely that the pathogen would have evolved to produce suchextensive diversity.The extensive diversity observed in the anthracnose pathogen in this study highlightsthe need for new Stylosanthes cultivars with different resistance to minimize potentialanthracnose damage to the limited range of cultivars available in Brazil, China and India.ReferencesChakraborty, S., Perrott R., Charchar M.J.D., Fernandes C.D., and Kelemu S. (1997).Biodiversity, epidemiology and virulence of Colletotrichum gloeosporioides. 11. Genetic andpathogenic diversity in Colletotrichum gloeoosporioides isolates from eight species ofStylosanthes guianensis.  Tropical grasslands 31:387-393.Chakraborty, S., Perrott R., Ellis. N. and Thomas M.R. (1999). New aggressiveColletotrichum gloeosporioides strains on Stylosanthes scabra detected by virulence andDNA analysis.   Plant Disease 83: 333-340.Guodao, L., Phaikaew C. and Stur W.W. (1997). Status of Stylosanthes development inother countries. II. Stylosanthes development and utilisation in China and Southeast Asia.Tropical Grasslands 31:460-466.Kelemu, S., Badel J.L., Moreno C.X., Miles J.W., Chakraborty S., Fernandes C.D. andCharchar M.J.d.A. (1997).  Biodiversity, epidemiology and virulence of Colletotrichumgloeosporioides. I. Genetic and pathogenic diversity in Colletotrichum gloeosporioidesisolates from Stylosanthes guianensis.  Tropical Grasslands 31:387-392.Kelemu, S., Skinner D.Z., Badel J.L., Moreno C.X., Rodriguez, M.X., Fernandes C.D.,Charchar M.J. and Chakraborty S. (1999). Genetic diversity in South AmericanColletotrichum gloeosporioides isolates from Stylosanthes guianensis, a tropical foragelegume. European Journal of Plant Pathology 105: 261-272.Miles, J.W. and Lascano C.E. (1997).  Status of Stylosanthes development in othercountries. I Stylosanthes development and utilisation in South America.  Tropical Grasslands.31: 454-459.Figure 1 - Genetic dissimilarity between 132 Colletotrichum gloeosporioides isolatescollected mainly from Australia, China, Colombia, Brazil and India.Group 1: 20 isolatesS.capitata, S.scabra,S.seabrana,S.fruticosaFromAustralia, Thailand,India, Colombia,BrazilGroup 2: 66 isolatesS.capitata,S.macrocephala,S.scabra,S.guianensisFromBrazilGroup 3: 19 isolatesS.capitataS.guianensisFromAustralia, Colombia,Africa, China, BrazilGroup 4: 27 isolatesS.guianensis,S.capitata, S.hamataFromBrazil, China,Colombia, Nigeria,The Philippines0.0           0.1             0.2            0.3            0.4             0.5            0.6            0.7            0.8Dissimilarity (%)

Genetic diversity in the anthracnose pathogen infecting Stylosanthes in Brazil, India and China

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Genetic diversity in the anthracnose pathogen infecting Stylosanthes in Brazil, India and China

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