A granular formulation of nomuraea rileyi farlow (samson) for the control of spodoptera frugiperda (lepidoptera: noctuidae).

Domenico Pavone, Mayri Díaz, Lesbia Trujillo and Blas Dorta

Domenico Pavone Maniscalco. M.Sc. in Agronomy, Universidad Central de Venezuela (UCV). Professor, Universidad de Carabobo (UC), Venezuela.

Mayri Díaz de Rienzo. Biologist and Graduate student, UCV, Venezuela.

Lesbia Trujillo Silva. Biologist, UCV, Venezuela.

Blas Dorta Dorta. Doctor in Biochemistry, Universidad Nacional de La Plata, Argentina. Professor, UCV, Venezuela. Address: Laboratorio de Procesos Fermentativos, Instituto de Biología Experimental, Facultad de Ciencias, UCV. Apartado 47114, Caracas 1050A, Venezuela. e-mail: bdorta@gmail.com

SUMMARY

A granular formulation of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson was evaluated against Spodoptera frugiperda (Lepidoptera: Noctuidae). The formulation consisted of 1mm particles of defatted corn germ (DCG) containing 10⁷ conidia/g. This preparation protected the conidia against UV radiation and killed 80% of S. frugiperda larvae in laboratory bioassays. It was shown that the fungus used DCG as a substrate for growth and sporulation, creating foci for further infection. This strategy has great potential for the formulation of fungal biocontrol agents, especially those with a high growth rate.

Una formulación granulada del hongo entomopatógeno Nomuraea rileyi (Farlow) Samson fue evaluada contra Spodoptera frugiperda (Lepidoptera: Noctuidae). La formulación consistió de partículas de 1mm de diámetro de germen desgrasado de maíz (GDM) inoculadas con 10⁷ conidias/g. La preparación protegió a las conidias de la radiación UV y eliminó al 80% de la población de larvas de S. frugiperda en bioensayos de laboratorio. Se demostró que el hongo es capaz de utilizar el GDM como un sustrato de crecimiento y esporulación creando nuevos focos de infección. Este tipo de estrategias posee gran potencial para la formulación de agentes fúngicos de biocontrol, especialmente aquellos con alta tasa de crecimiento.

RESUMO

Uma formulação granulada do fungo entomopatogênico Nomuraea rileyi (Farlow) Samson foi avaliada contra Spodoptera frugiperda (Lepidoptera: Noctuidae). A formulação consistiu de partículas de 1mm de diâmetro de germem desengordurado de milho (GDM) inoculadas com 10⁷ conidias/g. A preparação protegeu às conídias da radiação UV e eliminou 80% da população de larvas de S. frugiperda em bioensaios de laboratório. Demonstrou-se que o fungo é capaz de utilizar o GDM como um substrato de crescimento e esporulação criando novos focos de infecção. Este tipo de estratégias possui grande potencial para a formulação de agentes fúngicos de biocontrole, especialmente aqueles com alta taxa de crescimento.

KEYWORDS/ Biological Control / Biopesticide / Entomopathogenic Fungi / Zea mays /

Entomopathogenic fungi have great potential for integrated pest management programs due to their specificity, mode of action and ease of application. Nomuraea rileyi (Farlow) Samson is an entomopathogenic fungus found in several countries, including Brazil and Venezuela. This fungus attacks important caterpillar pests of soybean and corn such as Spodoptera frugiperda Smith (Lepidoptera: Noctuidae), causing epizootics (Ignoffo et al., 1976; Piñango et al., 2002). Environmental conditions (solar radiation, humidity, etc) greatly affect the microorganisms, decreasing their field viability and persistence. Current research has thus been focused on minimizing the effect of these conditions to increase fungus survival and effectiveness.

Biocontrol agent formulations are powerful tools for achieving this goal (Auld 1992; Goettel and Roberts, 1992; Pereira and Roberts, 1991; Rodham et al., 1999; Lacey et al., 2001). Liquid formulations with added oil improve fungus field performance (Prior et al., 1988; Bateman et al., 1993; Ibrahim et al., 1999; Luz et al., 1999; Maiga et al., 1999; Smith et al., 1999; De Courcy et al., 2000; Inyang et al., 2000) and additives such as chemical UV filters can protect fungi from UV radiation (Inglis et al., 1995; Leland et al., 2001).

This study describes the development of a granular formulation based on N. rileyi, which could function both as a growth culture medium and as an aid to fungal sporulation in the field, thus creating foci for further infection, leading to improved efficiency for the control of S. frugiperda.

N. rileyi isolate LPFIBE-3, supplied by the Centro Venezolano de Colecciones de Microorganismos (CVCM), is a strain originally obtained from a mummified field-collected larva of S. frugiperda in a corn plantation in Guárico State, Venezuela. The fungus was maintained on DYPA agar slants containing 5g dextrose, 1g peptone, 2g yeast extract, 1g NH₄NO₃, 1g K₂HPO₄, 0.5g MgSO₄.7H₂O, 0.01g FeCl₃.6H₂O and 16g agar per liter. S. frugiperda larvae were obtained from a population reared in the laboratory as described elsewhere (Parra, 1986).

N. rileyi was cultured in 500ml cylindrical screw-capped glass bottles of 10cm bore. Bottles containing 100ml of DYPA medium and allowed to solidify in a flat position were inoculated with 0.5ml of a conidia suspension of N. rileyi at a concentration of 10⁶ conidia/ml. Conidia were spread on the surface and the bottles were incubated at 25 ±2°C for two weeks, under continuous artificial light. After incubation, conidia were harvested by adding 100ml of sterile 0.1% Tween 80 in distilled water and shaking by hand. The concentration of conidia was determined in a Neubauer haemocytometer.

Granules consisted of defatted corn germ (DCG), supplied by Empresas Polar (Promasa), Turmero, Aragua State, Venezuela. This byproduct is an excellent solid substrate for culturing N. rileyi (Pavone, 2003). Autoclaved DCG was inoculated with N. rileyi conidia at a concentration of 10⁷ conidia/g dry matter, and the preparation’s water content was adjusted to 50% on a wet weight basis. The preparation was aseptically extruded through a cribbed plaque using a hand-operated mincing-type machine to obtain 2mm diameter filaments. These filaments were finally broken into 2-3mm-long fragments which were packed in glass columns of 2.5cm bore and aseptically air-dried. Filterd sterilized air was pumped through the columns at a rate of 0.3l·h^-1·g^-1 wet matter.

Water activity (a_w) of granules at different degrees of hydration (w_c) was measured at 25°C with a water activity analyzer Aqualab CX-2, (Decagon Devices Inc., Pullman, WA, USA). Water content was determined by an LJ-16 humidity analyzer (Mettler-Toledo AG, Greifensee, Switzerland).

Immediately after the drying process, 1g samples of dry granular formulation were placed in 30ml sterilized plastic containers and incubated until hydration at 25 ±2°C in a room saturated with water vapor under continuous artificial light. Incubations were carried out for 12 days, after which conidial yield was determined. Sporulated granules were re-suspended in 5ml 0.1% Tween 80/g of initial dry matter and conidia were counted as described above.

The granular formulation was exposed to UV radiation using a UVLMS-38 lamp (UVP^®; Ultra-violet Products, Upland, CA, USA). Three wavelengths were used in independent experiments: 254nm (UV-C), 302nm (UV-B) and 365nm (UV-A) at intensities of 250, 1600 and 2500μW·cm^-2, respectively, adjusted with a UVX radiometer (Ultra-violet Products, Upland, CA, USA). The granular formulation was exposed to UV radiation in open Petri dishes for 15min with periodic agitation.

Forty second instar S. frugiperda larvae per treatment were maintained individually in 30ml plastic containers. One gram of the granular formulation was placed in each container and larvae were fed on discs (2cm diameter) of Ricinus comunis L. leaves. Heat inactivated granules or alternatively leaf discs submerged in a suspension of 10⁷ conidia/ml were used as controls. The bioassay was checked daily and the number of dead larvae for each treatment was noted.

Mean lethal times 50 and 95 (LT₅₀ and LT₉₅) were estimated by Probit analysis using the Probit Analysis Program (Raymond, 1985). Differences between treatments were determined by comparing confidence levels given by the Probit analysis. The best fit isotherm curve was calculated using the software Curve Expert version 1.37.

The aim of the design of the granular formulation was to produce a solid culture medium for the promotion of sporulation of N. rileyi in the field. One of the most important requirements to accomplish this is to provide an adequate water supply to the granules. The water may come from rain, irrigation or, as in this study, water vapor from the atmosphere. The experiments were carried out at various relative humidity values in order to measure the sporulation response of N. rileyi on the granules. Water availability for the growth of the fungus on the granules is more dependent on water activity (a_w) than water content (w_c) per se, a_w being the relation between the vapor pressure of the granule-water mix and the vapor pressure of pure water. Under equilibrium conditions a_w/100= %RH. The water adsorption isotherm of the granular formulation, that is, the relation between w_c and a_w at a constant temperature, was determined with non-inoculated granules (Figure 1).

The relationship between w_c and a_w is best described by the Langmuir model (Langmuir, 1918), which was statistically validated by Fowler (1935). Data analysis led to w_c= 1/(-1.52+1.55·a_w^(-0.13)) to describe the isotherm (correlation coefficient= 0.99). A minimal water content (w_c= 30% on a wet-weight basis) is required to reach maximum water availability (a_w= 0.999). This value was reached at about 40h when the dry granules were incubated at 100% RH (Figure 2). Filamentous fungi require high levels of a_w for growth and sporulation on solid media (Dorta et al., 1990). An optimal a_w value of 0.977 for the growth of N. rileyi was determined in our laboratory (unpublished data), which corresponds to a w_c value of ~30%, according to the adsorption isotherm shown in Figure 1. Based on these results, it can be assumed that under conditions of 100% RH in the field, at least 40h are needed to reach the minimal w_c needed for growth and sporulation of N. rileyi on the granules.

However, as 100% RH is not always reached in the field, it is important to identify the ability of granules to absorb water at lower RH values. Water relations using saturated solutions are shown in Figure 1. RH conditions (11-100%) were characteristic of each saturated salt solution at equilibrium. Thus for each RH condition, the granules reached a constant w_c value whose magnitude agreed with the corresponding adsorption isotherm.

The results shown in Table I indicate that under different RH conditions granules absorb water until equilibrium is reached, which occurs sooner at lower RH values. At equilibrium, the granules stop adsorbing water; thus, at RH values lower than 97% granules will not be able to reach the minimal w_c (30% on a wet-weight basis) required to support the growth and sporulation of N. rileyi. However, at higher RH values (>97%) the water necessary for fungal growth is appropriately supplied.

The ability of N. rileyi to grow and sporulate on the formulated granules was demonstrated under appropriate conditions (100% RH; Figure 3). Sporulation begins on day 9, reaching a maximum yield of 6´10⁹ conidia/g dry matter on day 12. Taking into account the initial conidial concentration in the granules (10⁷ conidia/g dry matter), it was possible to increase the inoculation rate by 600 times using this method. Values of 6.5´10⁸ conidia/g were used in a granular formulation of M. anisopliae for the control of the lesser grain borer Rhyzopertha dominica (Batta, 2005) and a residual effect for both the granular and liquid formulations was reported. Figure 3 also shows the effect of sporulating granules on the mortality of S. frugiperda larvae. Indeed, after 9 days of hydration, incubated granules were able to cause 100% mortality.

The supplementation of granular formulations with carbon and nitrogen sources has been proposed to enhance sporulation of fungi, although the establishment of cost-benefit ratios is necessary before including these substances (Shah et al., 1999). In the present granular formulation, conidia production per gram was not significantly different from preparations based on GDC alone or those supplemented with sugar cane molasses and/or corn steep liquor (data not shown).

Granular formulations have been prepared with the fungi Arthrobotrys dactyloides and Verticillium chlamydosporium, the latter always having a less prolific growth than the former (Stirling and Smith, 1998). This fact has important field implications since granules may behave in two ways, as infection foci and as agents for the multiplication of the inoculum. N. rileyi is a slow-growing fungus requiring 10-12 days to complete growth and sporulation (Figure 3). This makes the growing process a slow one, increasing the probability of granule contamination. However, this strategy seems to be promising with faster-growing fungi such as Metarhizium anisopliae (Metschnikoff) Sorokin, Beauveria bassiana (Balsamo) Vuillemin and Trichoderma harzianum Rifai. Indeed, granular formulations of Trichoderma spp. have been prepared using vermiculite and wheatbran without aseptic conditions, allowing the proliferation of the biocontrol agent (Lewis and Lumsden, 2001).

Bioassays were carried out to evaluate the protective effect of the granules against UV radiation. Figure 4 and Table II show the effectiveness of the granular formulation for killing S. frugiperda larvae. The results indicate that the granular formulation was able to reduce 75% of the larval population, compared to unformulated liquid conidia. In this case, conidia were spread on the surface of R. communis leaf discs promoting contact with the larvae. In addition, a large number of conidia were ingested by the larvae as they fed on leaf discs. It is important to emphasize that in this case the granular formulation was not yet colonized by the fungus and mortality was therefore due exclusively to conidia on the surface of the granule. Additionally, most conidia were inside the granule matrix, and thus not in direct contact with the larvae. Thus, the number of accessible conidia in the granular formulation was less than on the leaf discs coming from the aqueous formulation. Granular formulations of B. bassiana (Maniania, 1993) and M. anisopliae (Ekesi et al., 2005) have been evaluated. These formulations were more efficient than spray applications of aqueous and oily aqueous formulations probably due to their greater persistence in the field.

LT₅₀ and LT₉₅ were not significantly different between granules exposed and not exposed to UV radiation (Figure 4; Table II). These results point to the protective effect of the formulation on the N. rileyi conidia from UV radiation. The irregular topography of the granule could act as a physical barrier to UV radiation; as a result, most conidia should be protected. However, this experiment did not determine how many conidia on the surface of the granule were affected by UV radiation, which depends on the surface portion exposed to UV radiation. It is clear that the conidia inside the granules should remain alive, because they were not exposed to UV radiation. It is also important to emphasize that the use of granular formulations based on CDG may have certain limitations, since mycotoxigenic fungus such as Aspergillus flavus Link: Fries, commonly found on corn fields, could also proliferate on this substrate (Lewis, 2001). The importance of determining the impact of granules on corn mycotoxin levels is obvious. The high capacity of the granules to cause mortality of S. frugiperda, the generation of infective foci and the UV-protection exerted show the potential of this granular formulation of N. rileyi for the biocontrol of this insect pest. It is important to emphasize, however, the importance of the implementation of field trials to probe the effectiveness and safety of the formulation under these conditions, which is the focus of our current research.

The authors thank Gilberto Payares for correcting the English and Ronaldo Toma for translating the abstract into Portuguese. This research was supported by a fellowship to Domenico Pavone and by Grant Nº S1-2001000968, both from the Venezuelan Research Foundation (FONACIT).

1. Auld B (1992) Mass production, formulation and application of fungi as biocontrol agents. In Lomer CJ, Prior C (Eds.) Biological Control of Locusts and Grasshoppers. CABI - International Institute of Tropical Agriculture. Ascot, UK. p 219.        [ Links ]

2. Bateman R, Carey M, Moore D, Prior C (1993) The enhanced infectivity of Metarhizium flavoviridae in oil formulations to desert locusts at low humidity. Ann. Appl. Biol. 112: 145-152.        [ Links ]

3. Batta Y (2005) Control of the lesser grain borer (Ryzopertha dominica (F.), Coleoptera: Bostrichidae) by treatments with residual formulations of Metarhizium anisopliae (Metschnikoff) Sorokin (Deuteromicotina: Hyphomycetes). J. Stored Prod. Res. 41: 221-229.        [ Links ]

4. Connick W, Daigle D, Pepperman A, Hebbar K, Lumsden R, Anderson T, Sands D (1998) Preparation of stable, granular formulations containing Fusarium oxysporum pathogenic to narcotic plants. Biol. Contr. 13: 79-84.        [ Links ]

5. De Courcy W, Edmondson R, Gill G (2000) The potential of some adjuvants in promoting infection with Verticillium lecanii: Laboratory bioassay with Myzus persicae. Ann. Appl. Biol. 137: 337-345.        [ Links ]

6. Dorta B, Bosch A, Arcas JA, Ertola R (1990) High level of sporulation of Metarhizium anisopliae in a medium containing by-products. Appl. Microbiol. Biotechnol. 33: 712-715.        [ Links ]

7. Ekesi S, Maniania N, Mohamed S, Lux S (2005) Effect of soil application of different formulations of Metarhizium anisopliae on African tephritid fruit flies and their associated endoparasitoids. Biol. Contr. 35: 83-91.        [ Links ]

8. Fowler RH (1935) A statistical derivation of the Langmuir adsorption isotherm. Proc. Cambridge Phil. Soc. Math. Phys. 31: 260-280.        [ Links ]

9. Goettel M, Roberts DW (1992) Mass production, formulation and field application of entomopathogenic fungi. In Lomer CJ, Prior C (Eds.) Biological control of locusts and grasshoppers. CABI / International Institute of Tropical Agriculture. Ascot, UK. p 230.        [ Links ]

10. Hua L, Feng MG (2003) New use of broomcorn millets for production of granular culture of aphid-pathogenic fungus Pandora neoaphidis for high sporulation potential and infectivity to Myzus persicae. FEMS Microbiol. Lett. 227: 311-317.        [ Links ]

11. Ibrahim L, Butt T, Beckett A, Clark S (1999) The germination of oil-formulated conidia of the insect pathogen, Metarhizium anisopliae. Mycol. Res. 103: 901-907.        [ Links ]

12. Ignoffo C, Marston N, Hostetter D, Puttler B, Bell J (1976) Natural and induced epizootics of Nomuraea rileyi in soybean caterpillars. J. Invert. Pathol. 27: 191-198.        [ Links ]

13. Inglis G, Goettel M, Johnson D (1995) Influence of ultraviolet light protectants on persistence of the entomopathogenic fungus, Beauveria bassiana. Biol. Contr. 5: 581-590.        [ Links ]

14. Inyang E, McCartney H, Oyejola B, Ibrahim L, Pye B, Archer S, Butt T (2000) Effect of formulation, application and rain on the persistence of the entomogenous fungus Metarhizium anisopliae on oilseed rape. Mycol. Res. 104: 653-661.        [ Links ]

15. Lacey L, Frutos R, Kaya H, Vail P (2001) Insect pathogens as biocontrol agents: Do they have a future?. Biol. Contr. 21: 230-248.        [ Links ]

16. Langmuir I (1918) The adsorption of gases on plane surfaces of glass, mica and platinum. J. Am. Chem. Soc. 40: 1361-1368.        [ Links ]

17. Leland JE (2001) Environmental-stress Tolerance Formulations of Metarhizium anisopliae var. acridum for Control of the African Desert Locust (Schisticerca gregaria). Thesis. Virginia Polytechnic Institute and State University. http://scholar.lib.vt.edu/theses/available/etd-12052001-115455/unrestricted/ JLelandDissertation.PDF (Cons. 10/11/2005).        [ Links ]

18. Leland JE, Mullins D, Smits N, Fargues J, Warren H, Larry V (2001) Coating Metarhizium anisopliae var. acridum with water-soluble lignins for enhanced UVB-protection and effects on virulence to Schistocerca americana (Drury). http://esa.confex.com/esa/2001/techprogram/paper_3552.htm (Cons. 06/01/2002).        [ Links ]

19. Lewis L (2001) Development of Alternative Carriers for Use of Beauveria bassiana in Ostrinia nubilalis Suppression on Corn. Leopold Center Progress Report, Vol. 10. www.ag.iastate.edu/centers/leopold/pubinfo/2001cpr/99-75.pdf (Cons. 11/15/2002).        [ Links ]

20. Lewis J, Larkin R (1998) Formulation of the biocontrol fungus Cladorrhinum foecundissimum to reduce damping-off diseases caused by Rhizoctonia solani and Pythium ultimum. Biol. Contr. 12: 182-190.        [ Links ]

21. Lewis J, Lumsden R (2001) Biocontrol of damping-off of greenhouse-grown crops caused by Rhizoctonia solani with a formulation of Trichoderma spp. Crop Protect. 20: 49-56.        [ Links ]

22. Luz C, Silva I, Magalhães B, Cordeiro C, Tigano M (1999) Control of Triatoma infestans (Klug) (Reduviidae: Triatominae) with Beauveria bassiana (Bals.) Vuill.: Preliminary assays on formulation and application in the field. Anais Soc. Entomol. Bras. 28: 101-110.        [ Links ]

23. Maiga I, Douro O, Lomer C, Langewald J (1999) Utilisation de Metarhizium avoviride Gams & Rozypal contre les sauteriaux dans des essais participatifs en milieu paysan au Niger. Insect Sci. Appl. 18: 279-284.        [ Links ]

24. Maniania NK (1993) Effectiveness of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuill. for control of the stem borer Chilo partulleus (Swinhoe) in maize in Kenya. Crop Protect. 12: 601-604.        [ Links ]

25. Parra JR (1986) Criação de insetos para estudos com patógenos. In Alves SB (Ed.) Controle Microbiano de Insetos. 1^st ed. Manole. São Paulo, Brazil. pp. 348-372.        [ Links ]

26. Pavone D (2003) Formulación de un Bioinsecticida con Base en el Hongo Nomuraea rileyi (Farlow) Samson para el Control de Spodoptera frugiperda Smith (Lepidoptera: Noctuidae). Thesis. Universidad Central de Venezuela. Caracas, Venezuela. 147 pp.        [ Links ]

27. Pereira R, Roberts D (1991) Alginate and cornstarch mycelial formulations of entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae. J. Econ. Entomol. 84: 1657-1661.        [ Links ]

28. Piñango L, Arnal E, Rodríguez B, Guerrero I, Pérez H, Ramos F (2002) Influencia de diferentes prácticas de manejo sobre la fluctuación poblacional del gusano cogollero (Spodoptera frugiperda) en el cultivo del maíz. In Mem. VI Festival Nacional del Maíz y VI Jornada Científica Nacional del Maíz, (11/20-23/2002). Facultad de Agronomía, Universidad Central de Venezuela. Maracay, Venezuela.        [ Links ]

29. Prior C, Jollands P, Le Patourel G (1988) Infectivity of oil and water formulations of Beauveria bassiana (Deuteromicotina: Hyphomycetes) to the cocoa weevil pest Pantorhytes plutus (Coleoptera: Curculionidae). J. Invert. Pathol. 52: 66-72.        [ Links ]

30. Raymond M (1985) Présentation d’un programme d’analyse log-probit pour micro-ordinateur. Cahiers ORSTOM. Sér. Entomol. Méd.t Parasitol. 22: 117-121.        [ Links ]

31. Rodham D, Wang Y, Cantwell J, Winn P, Foundling J (1999) Formulating microbial biocontrol agents. Pesticide Sci. 55: 340-342.        [ Links ]

32. Schwarz M (1995) Metarhizium anisopliae for soil pest control. In Hall FR, Barry JW (Eds.) Biorational Pest Control Agents: Formulation and Delivery. American Chemical Society. Washington DC, USA. pp 183-196.        [ Links ]

33. Shah P, Aebi M, Tuor U (1999) Production factors involved in the formulation of Erynia neoaphidis as alginate granules. Biocontr. Sci. Technol. 9: 19-28.        [ Links ]

34. Shah P, Aebi M, Tuor U (2000) Drying and storage procedures for formulated and unformulated mycelia of the aphid-pathogenic fungus Erynia neoaphidis. Mycol. Res. 104: 440-446.        [ Links ]

35. Smith S, Moore D, Karanja L, Chandi E (1999) Formulation of vegetable fat pellets with pheromone and Beauveria bassiana to control the larger grain borer, Prostephanus truncatus (Horn). Pesticide Sci. 55: 711-718.        [ Links ]

36. Stirling G, Smith L (1998) Field tests of formulated products containing either Verticillium lecanii or Arthrobotrys dactyloides for biological control of root-knot nematodes. Biol. Contr. 11: 231-239.        [ Links ]

37. Walker HL, Connick WJ (1983) Sodium alginate production and formulation of mycoherbicide. Weed Sci. 31: 333-338.        [ Links ] ]]> 1 1992 219 2 1993 112 145-152 3 2005 41 221-229 4 1998 13 79-84 5 2000 137 337-345 6 1990 33 712-715 7 2005 35 83-91 8 1935 31 260-280 9 1992 230 10 2003 227 311-317 11 1999 103 901-907 12 1976 27 191-198 13 1995 5 581-590 14 2000 104 653-661 15 2001 21 230-248 16 1918 40 1361-1368 17 2001 18 2001 19 2001 10 20 1998 12 182-190 21 2001 20 49-56 22 1999 28 101-110 23 1999 18 279-284 24 1993 12 601-604 25 1986 1st 348-372 26 2003 147 27 1991 84 1657-1661 28 2002 2002 Maracay 29 1988 52 66-72 30 1985 22 117-121 31 1999 55 340-342 32 1995 183-196 33 1999 9 19-28 34 2000 104 440-446 35 1999 55 711-718 36 1998 11 231-239 37 1983 31 333-338