Research Journal of Recent Science s ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 110 - 115 (201 3 ) Res.J. Recent .Sci. International Science Congress Association 110 Genetic variability of Macrophomina phaseolina Affecting Sesame : phenotypic traits, RAPD markers and interaction with the Crop Martínez - Hilders Andrea, Mendoza Yuraima, Peraza Dasybel and Laurentin Hernán Departamento de Ciencias Biológicas, Decanato de Agronomía, Universidad Centroccidental Lisandro Alvarado, Barquisimeto, VENEZUELA Available online at: www.isca.in Received 0 4 th August 2012, revised 26 th January 201 3 , accepted 07 th February 201 3 Abstract Macrophomin a phaseolina is a fungus which affects more than 300 cultivated species. It is one of the most important biotic stresses on sesame (Sesamum indicum L.). A successful control strategy, especially plant resistance management, depends on comprehensive knowled ge about genetic variability for both fungus and plant. To evaluate genetic diversity of M. phaseolina affecting sesame in the most important crop production region of Venezuela, seven isolates were characterized by means of phenotypic traits, and RAPD mar kers. Four of these isolates were used for evaluating the interaction with four sesame genotypes in two ways: interaction in vivo by inoculation, and effect of root and stem extracts on fungus growth. Variability for growth velocity (optical density ranged between 1.69 to 2.32 at 96 hours of growing ) (P≤0.05) and microsclerotia production (18 - 56 in 100 μL) (P≤0.05), was observed. Ten primers used were able to amplify the DNA, generating 81 bands (100% polymorphic). Ordination of the seven isolates by means of principal coordinates analysis based on R APD did not show a consistent relationship with phenotypic attributes or geographical origin. After inoculation, length lesion produced by the four isolates did not show statistical differences, but germination percentage did (P≤0.01). One of the fungus is olates reduced up to almost 70% average germination of the four sesame genotypes. Mycelial growth of four isolates was inhibited in 17 – 32 % as compared to the control. These results indicate it is difficult to manage charcoal rot by means of obtaining re sistant cultivars because of the fungus variability found in all the levels evaluated.. Keywords : DNA, charcoal rot, sesame, inoculation, morphological traits . Introduction Charcoal rot is a disease caused by the fungus Macrophomina phaseolina (Tassi) Goid. On more than 300 cultivated species, causing obstruction at root tissue 1 . It is an important threat to sesame ( Sesamum indicum L.) production in Venezuela 2 . Sesame season in Venezuela (November - April) is characterized by high temperatures and dry soils because of lack of rain, which are favorable conditions for charcoal rot 3 . In Venezuela, losses in sesame due to this fungus have been evaluated, resulting up to 65% of seed weigh t reduction for affected plants 4 . M. phaseolina survives in the soil by means of sclerotia, which have been quantified in Venezuelan soils of sesame production areas up to 200 g - 1 of soil 5 . Suitable identification of plant pathogens is a key factor for establis hing disease control strategies 6 , for that, genetic and morpholog ical characterization is needed. Characterization of individuals in a population is defined as the description based o n phenotype or molecular traits 7 , and both strategies are complementary accord ing to the information provided 8 . One of the strategies for controlling the pathogen is the use of genetic resistance, but for using it, it is necessary a wide knowledge about genetic variability for both Macrophomina phaseolina and Sesamum indicum . Furthermore, knowledge about interaction between isolates of the f ungus and sesame cultivars is required. Interaction host - pathogen could be studied in vivo and in vitro . Studies of interaction in vivo need an efficient inoculation protocol to ensure the pathogen reaches to the host, and the host is able to respond. Stud ies of interaction sesame - M. phaseolina has been previously reported (e.g. 9,10 ). For in vitro studies, an approach used is to confront the pathogen to plant extracts and to evaluate the effect of this extract on pathogen growth. If extracts have some bioch emical compounds which are toxic to the pathogen, the fungus will inhibit the growth. This kind of evaluation could have a huge potential. Number of plant species is about 500,000 but only few of them have been studied according to antimicrobial activity o f extracts 11 . Effect of cultivated plant extracts (sunflower) on M. phaseolina have been reported (e.g. 12 ), but never sesame extracts effect on this fungus has been evaluated. The objective of this investigation was to evaluate genetic diversity of Macrop homina phaseolina coming from Venezuelan sesame production, in four different ways by means of: a. phenotypic attributes, b. molecular markers (RAPD), c. ability to cause disease on four sesame genotypes, d. growth with sesame extracts. Material and Meth ods Fungus isolates : Tissue presenting the disease collected from plants in the field was obtained to get seven isolates ( t able 1). Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 1 10 - 115 (201 3 ) Res.J.Recent.Sci . International Science Congress Association 111 Table - 1 Location of collected material at Venezuelan sesame production area to get seven isolates of M. phaseolina Isolate name Collecting place Latitude of collecting place Longitude of collecting place Altitude of collecting place (m.a.s.l.) Collected material 2 - 2010 Acequioncito 9º12´41,01´´ N 68º56´47,8´´ W 100 Sesame tissue C3 - 2010 In between Chorrerones and El Ají 9º07 ´43,18´´ N 69º01´44,54´´ W 114 Sesame tissue 2 - 2011 Road V 9º18´10,4´´ N 69º07´13,9´´ W 133 Bean tissue 30 - 2011 Road V 9º18´10,4´´ N 69º07´13,9´´ W 133 Sesame tissue 36 - 2011 El Gateao 9º 07´ 38,3´´ N 68º53´ 19,8´´ W 90 Sesame tissue 37 - 2011 El Playón 9 º 06´34,1´´ N 69º 02´ 31,3´´ W 96 Sesame tissue 41 - 2011 Camino 8 9º09´4,9´´N 68º54´06´´W 100 Sesame tissue Tissue was incubated in Petri dishes containing potato dextrose agar to promote mycelia growth, which was transferred many times to other Petri d ish until getting typical, clean and pure Macrophomina phaseolina mycelia, in the same way used by Csöndes I. and et al 13 . Seven isolates were obtained by this methodology Phenotypic characterization : Two attributes were evaluated for the phenotypic chara cterization: growth velocity and number of microsclerotia. For number of microsclerotia, a completely randomized design with six replications was used, where the experimental unit was a Petri dish containing the fungus growing for two weeks. One gram of me dium potato dextrose agar containing mycelium and microsclerotia was macerated in 10 mL distilled water From this volume, 100 µL were taken to count the number of microsclerotia; this was repeated 5 times, therefore the reported values for each experimenta l unit is the average of the 5 counted volumes. For growth velocity, 50 microsclerotia were obtained from a 14 days - old culture, and they were placed in an individual well of 96 - well microplates used for ELISA, containing 200 µL of potato dextrose broth. I t was repeated 64 times for each fungus isolate. Optical density of each individual well was measured each 12 hours by means of a spectrophotometer Multiskan FC (Thermo, Finland) at 450 nm. The more is the optical density, the more is the interference in e ach well caused by mycelia growth. Molecular characterization : One hundred fifty milligrams of mycelium of each isolate growing in potato dextrose broth were used for DNA extraction wi th Dellaporta extraction buffer 14 , containing 10 µL of mercapthoethano l and 10 µL of proteinase K (20 mg mL - 1 ). The homogenates were incubated for 20 m at 65°C, followed by addition of 500 µL of phenol:chlorophorm:isoamylic alcohol (25:24:1). Phases were separated by centrifugation for 10 min at 14000 rpm. Isopropanol (750 µ L), and ammonium acetate 5 M (5 µL) were added to the aqueous phase. After 30 min samples were centrifuged at 14000 rpm for 10 min, pellet were washed twice with 70% ethanol, dried and dissolved in 50 µL of TE buffer. Additional step of cleaning with ammon ium acetate and RNAse was done. DNA quantity and quality was determined by electrophoresis in a 0,8% agarose gel with lambda DNA standard. RAPD analysis for each combination isolate - primer was performed using 20 ng of DNA, 4 µL of PCR 5X buffer, 2 µL of Mg Cl 2 25 mM, 4 µL of dNTPs 10 mM, 0,8 µL of gelatine 0,025% (w/v), 1 µL 2mM of one of 10 primers (OPA02, OPA03, OPA04, OPA05,OPA07,OPA09, OPA13, OPC04, OPC06 and OPC08), 1 U of Taq polymerase, in a final volume of 20 µL. The thermocycler program consisted of a first step at 93°C for 2 min, followed by 45 cycles consisting in 93°C for 1 min, 36°C for 1 min and 72°C for 1 min. Final step consisted in 72°C for 5 min. PCR products were resolved in agarose gel 1.4% w/v for 60 min at 60 V. Gels were stained in ethi dium bromide and washed in water for visualizing band under UV light. Inoculation on sesame plantlets : Plastic trays (10x19x5 cm) were filled of a mixture of sterilized substrate with a content of a Petri dish containing the fungus for two weeks on potat o dextrose agar. Four trays were used for each of 4 isolates (2 - 2011, 36 - 2011, 37 - 2011 and 41 - 2011). For each tray, four sections were defined to sow 10 seed of each of 4 sesame cultivars (Maporal, UCLA295, 43x32 and India7), resulting in a split plot desi gn with four replication. Isolates were the main plot, and sesame cultivar the subplot. After sowing, substrate was irrigated with 20 mL of distilled water. On each experimental unit the variables measured were germination percentage and length lesion on t he stem. Sesame extracts on fungus growth : Fifty seeds of each sesame cultivar (Maporal, UCLA295, 43x32 and India7) were germinated in a sterilized substrate. Three weeks later, roots were separated from stem. Each mass of root was homogenized in ethanol 80% in a ratio of 1 g of mass to 5 mL of ethanol, and kept at room temperature for 16 hours. Afterwards they were filtered and kept at 4°C until bioassays were established. Bioassays were performed in 96 - wells microplates. Each well was filled with 200 µL of root extract; as control wells were filled with 200 µL of ethanol (without extracts). After 24 h (when ethanol was evaporated), 200 µL of potato dextrose broth containing 50 microsclerotia were poured in each well, including the control. This procedure was repeated eight times (eight wells) for each combination root extract – fungus isolate, for each sesame genotype. Fungus isolates used were 2 - 2011, 36 - 2011, 37 - 2011 and 41 - 2011. Optical density at 450 nm was recorded when bioassay was established, and e ach 12 hours during 120 hours. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 1 10 - 115 (201 3 ) Res.J.Recent.Sci . International Science Congress Association 112 Results and Discussion Broad variation among fungus isolates was identified. Microsclerotia production ranged between 18 and 53, identifying the mean test that 2 - 2010, 30 - 2011 and C3 - 2010 had the highest production of micros clerotia, almost 3 - times more than 2 - 2011 ( table 2). Growth velocity also displayed variation among isolates ( figure 1), and according Tukey test (data not shown) three groups were formed: the first one with the lowest velocity, conformed by C3 - 2010, 2 - 201 1 and 37 - 2011, the second one with an intermediate growth velocity conformed by 30 - 2011, 36 - 2011 and 41 - 2011, and the third one with the highest growth velocity represented by 2 - 2010. Optical density as indicator of fungus growth shows the typical form of the curve in the figure 1: low growth the first 24 h, exponential growth from 24 h to 60 h, and decrease of growth rate from 60 h, therefore optical density measure is a reliable way to record M. phaseolina growth through the time. Other authors report gro wth velocity of M. phaseolina , but they did it in the conventional way (e.g. 15, 16 ), it is not possible to compare them to our results. Both microsclerotia production and growth velocity are attributes related to aggressiveness of pathogen isolates, therefo re isolate 2 - 2010 seems to be the most aggressive because of the ability to grow quickly and produce high amount of inoculum. On the other hand, 2 - 2011 has the lowest aggressiveness, it grows slowly and produce low amount of microsclerotia. Table - 2 Numbe r of microsclerotia of seven isolates of M. phaseolina counted in 100 µL of water after macerating 1 g of fungus tissue growing on potato dextrose agar during 2 weeks Fungus isolate Number of microsclerotia 2 - 2010 56.17 a 30 - 2011 53.67 a C3 - 2010 53.00 a 36 - 2011 41.00 b 37 - 2011 23.77 c 41 - 2011 20.70 cd 2 - 2011 18.10 d Means followed by the same letter are not statistically different (P.05) according to Tukey test Figure - 1 Growth velocity for seven isolates of M. phas eolina recorded in 96 - wells microplates. The lowest and intermediate line indicate growth as average on the isolates which did not show statistical differences in growth Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 1 10 - 115 (201 3 ) Res.J.Recent.Sci . International Science Congress Association 113 Figure - 2 Biplot from principal coordinates analysis showing ordination of seven M acrophomina phaseolina isolates based on 81 RAPD bands Figure - 3 Optical density in wells of microplates as indicator of growth velocity for four isolates of M. phaseolina growing with sesame root extracts Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 1 10 - 115 (201 3 ) Res.J.Recent.Sci . International Science Congress Association 114 Molecular charact erization based on RAPD generated 81 bands (100% polymorphic). Seventy five percent of the bands had a size range between 150 and 1000 base pairs (bp). Ratio number of bands per primer resulted in 8.1. A previous characterization of the fungus found a rati o of 3.36 17 . Primer OPA13 was reported as the one which amplified on all M. phaseolina isolates 18 , however it did not occur for the seven isolates evaluated in this research. This is evidence that the value of some primers could vary depending on the popul ation used, they are not necessarily valuable through all the population in a species. Figure 2 shows ordination of isolates by means of principal coordinates analysis. There was not a consistent relationship between grouping based on RAPD and phenotypic t raits, or geographical origin. Furthermore, RAPD did not discriminate the isolate coming from bean. However there was a trend to separate isolates according to the margin of Acarigua River (main river of the zone) from where they were collected. There is l ittle evidence which supports independent evolution of isolates, on the contrary, it seems there is a constant exchange of microsclerotia through sesame production area, which is consistent with the constant seed exchange among farmers. Effect of the fun gus isolates on four sesame genotypes was not different in length lesion, it ranged between 10.3 to 18.5 mm but they did not show significant differences (P.05); however, they were different when germination percentage was evaluated ( table 3). When isola te 2 - 2010 was inoculated, germination resulted in only 30% as compared to the control. This result is consistent with the phenotypic attributes, which identifying isolate 2 - 2010 as the most aggressive. Variability among isolates of M. phaseolina on sesame seed germination has been previously reported 5. Table - 3 Length lesion and germination percentage caused by four isolates of M. phaseolina (values were averaged on the damage of four sesame genotypes) Fungus isolate Length lesion (mm) Germination percenta ge 36 - 2011 10.3 a 85 a 37 - 2011 18.5 a 76 a 41 - 2011 14.1 a 72 a 2 - 2010 13.7 a 32 b Means followed by the same letter are not statistically different (P.05) according to Tukey test Sesame root extracts inhibited growth of the four isolates of M. phas eolina . Figure 3 show the optical density for each isolate during 120 h averaged on the four sesame cultivars used. The behavior for the four isolates was very similar, showing inhibition since hour 72; however 2 - 2010 was more inhibited than 37 - 2011; 2 - 201 0 grew 68% as compared to the control, whereas 37 - 2011 did it 83%. There is no previous reports about effect of sesame root extracts on M. phaseolina , however, there is a previous report of effect of root extract of another cultivated plant (sunflower) on this fungus; these authors found a strong inhibitory effect 12. Conclusion Macrophomina phaseolina presented a broad genetic variation in the Venezuelan sesame production area in all the levels which were evaluated: phenotypic traits, molecular markers, e ffect on sesame cultivars and response to sesame root extracts. These results indicate it is difficult to manage charcoal rot by means of obtaining resistant cultivars because of the fungus variability. For generating this kind of cultivars could be necess ary stratification of the area according to genetic variability of the fungus. Acknowledgement Authors want to express the acknowledgement to Consejo de Desarrollo Científico, Humanístico y Tecnológico (C.D.C.H.T.) of Universidad Centroccidental Lisandro Alvarado and to Internactional Foundation Sciences (IFS) for financial support of the project 033 - AG - 2009 and IFS C/4408 - 1 respectively, from which resulted this paper. References 1. Wyllie T. Charcoal rot, In: Compendium of soybean diseases. Third edition. J.B. Sinclair and P.A. Backman (eds). Pages 30 - 33. American Phytopathological Society, St. Paul, MN, USA , (1989) 2. Pineda J. Enfermedades del ajonjolí: algunas medidas de control. II Curso sobre producción de ajonjolí, soya y otras leguminosas , 114 pp. ASOP ORTUGUESA, UCLA and FONAIAP, Venezuela (2002) 3. Odvody G. and Dunkle L. , Charcoal stalk rot of sorghum: effect of environment in host - parasite relations , Phytopathology, 126 , 343 - 352 (1979) 4. 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