Rev. FCA UNCUYO. 2016. XX(X): XXX-XXX. ISSN impreso 0370-4661. ISSN (en línea) 1853-8665.

First record of coronata (), a possible health significance associated with tomato crops in Argentina

Primer registro de Diploscapter coronata (Rhabditida), un posible nematodo de importancia sanitaria asociado a cultivos de tomate en Argentina

Augusto Salas 1, José Matías Rusconi 1, Nora Camino 1, 2, Daiana Eliceche 1, María Fernanda Achinelly 1, 3

Originales: Recepción: 21/03/2016 - Aceptación: 08/10/2016 Nota científica

Abstract

Diploscapter coronata is a free-living soil bacterial-feeding nematode found in compost, sewage or agricultural soil and as a facultative parasite of insects and verte- brates, even humans. The clinical symptoms include epigastric tenderness, diarrhea, crampy abdominal pain, weakness and nauseas. Also, they have been considered as potential carriers of bacteria pathogenic to the surface of preharvest fruits and vegetables in contact with soil. In this note, we reported the presence of D. coronata in the framework

Argentina. Soil samples taken from tomato growing (Lycopersicon esculentum) were processedof diverse soil in thenematodes laboratory samplings by the centrifugationin orchards of Abasto method, town, while Buenos collected Aires roots province, were observed directly under stereomicroscope in order to isolate . Specimens presence of D. coronata in agricultural soil and in association with root galls, caused by thewere plant-parasitic identified by morphologicalnematode, and morphometric aberrans. Females characteristics. were the Resultsonly isolated showed stage. the The detection of this nematode in greenhouses where dogs, cats and poultry live together without any health control highlights the importance of applying proper hygiene measures during agricultural practices to avoid contamination of fruits and vegetables and prevent

Diploscapter genus with the species D. coronata in Argentina. infections in domestic and humans. This report constitutes the first record of the Keywords Diploscapter coronata • Lycopersicon esculentum • soil nematodes • facultative parasite • root galls • 1 Centro de Estudios Parasitológicos y de Vectores, (CEPAVE). CONICET, Universidad Nacional de la Plata, Argentina. [email protected] 2 CEPAVE Miembro de la carrera de Investigación de la Comisión de

(CCT, La Plata, CONICET). 3 CEPAVEInvestigaciones Miembro Científicas de la carrera (CIC) de de Investigación Buenos Aires. de CONICET A. Salas et al.

Resumen

Diploscapter coronata es un nematodo de vida libre que habita el suelo y se alimenta de bacterias. Se ha encontrado en compost, alcantarillas o suelos agrícolas y como parásito facultativo de insectos y vertebrados, incluyendo al hombre. Los síntomas clínicos incluyen: sensibilidad epigástrica, diarrea, dolor abdominal y náuseas. Incluso, han sido considerados potenciales portadores de bacterias patogénicas asociadas con la se reporta la presencia de D. coronata en el marco de diversos muestreos de nematodos superficie de frutas y vegetales pre-cosechadas en contacto con el suelo. En esta nota, Las muestras de suelo tomadas de cultivo de tomate (Lycopersicon esculentum) se procesaronde suelo, en en huertas el laboratorio de la localidad mediante de elAbasto, método provincia de centrifugación, de Buenos mientrasAires, Argentina. que las raíces recolectadas fueron observadas directamente bajo microscopio estereoscópico - terísticas morfológicas y morfométricas. Los resultados mostraron la presencia de D. coronatacon el fin en de suelos aislar agrícolas los nematodos. y en agallas Los especímenesradicales causadas fueron por identificados el nematodo por parásito carac de plantas Nacobbus aberrans. Las hembras fueron el único estadio aislado. La detección de este nematodo en invernaderos donde gatos, perros y aves de corral viven juntos sin ningún control sanitario, realza la importancia de adoptar medidas apropiadas de higiene durante las prácticas agrícolas para evitar la contaminación de frutas y verduras y prevenir infecciones en animales domésticos y el hombre. Este informe constituye el primer registro del género Diploscapter con la especie D. coronata en Argentina.

Palabras claves Diploscapter coronata • Lycopersicon esculentum • Nacobbus aberrans nematodos de suelo • parásito facultativo • agallas radicales • Introduction

Rhabditid nematodes are very abundant with clinical signs of the gastrointestinal or in all types of soil and sediments of fresh- genitourinary systems (5, 6, 10, 12, 13). In water bodies and play important ecological addition, their potential as vectors of food- roles mainly as primary consumers, but borne pathogenic bacteria, to the surface of also as parasites (1). preharvest fruits and vegetables in contact The genus Diploscapter Cobb, 1913, with soil has been determined (9). includes free-living bacterivorous nematodes In South-America, the presence of characterized by an unusual head with this nematode has been reported for hook-like appendages and membrane-like lateral lips (4). Among them Diploscapter In this work, we extend this distri- coronata (Cobb, 1893) Cobb, 1913 is a cosmo- bution,Venezuela, with Brazil, the Peru report and of Paraguay Diploscapter (4). politan rhabditid nematode obtained from coronata in the framework of a nematode decaying banana roots in Fiji Island (7), and soil study of different crop areas including later from agricultural soils, compost, sewage tomato samplings in Argentina, consti- and as a facultative parasite of vertebrates (cats, dogs, snakes, tigers), even humans, species for this country. tuting the first report of the genus and Materials and methods Diploscapter coronata has been collected previously from soil and Specimens were isolated during 2015, decaying plant material in association in a survey of Phytophagous nematodes with forest and horticultural crops, and associated with tomato crops, from four as a facultative parasite of animals, even orchards located in the Abasto town humans (1, 12). However, morphometric data of these nematode populations are province, Argentina. Twenty sub-samples scarce in the world (table 1, page XXX). of(34°56'35" 20 cm deep S 58°5'30" were W), taken Buenos to form Aires a In this work, females were isolated from composed sample from each site. Roots agricultural soil and in association with of tomato plants with signs of damage by root galls, caused by the plant-parasitic phytonematodes were also collected. nematode, Nacobbus aberrans (Thorne, In the laboratory, each composed 1935) Thorne & Allen, 1944. Specimens sample was homogenized and sieved. were observed inside and over the surface Nematodes were isolated from soil by of galls. Phytophagous nematodes cause sugar centrifugation method (100-cm3 soil wounds in the roots favoring the penetration by each site) for 5 minutes at 2700 rpm, of other pathogens, and are mainly respon- and from roots by examination and sible for major damage to plants (2, 11). dissection under stereoscopic microscope Diploscapter coronata (10). Characterization was carried out could be one of the reasons of the presence of by a morphometric and morphological thisBacterial-feeding nematode in association of with root galls analysis, using a light microscope, of tomato crops, which may be attracted by bacteria of the rhizosphere being able Measurements were given in micrometers. to penetrate plant tissues through wounds Photographsfollowing specific were taken bibliography using an Olympus (4). produced by plant parasitic nematodes. DP-71 microscope camera. Males of D. coronata were not observed in our study. The causes are unknown, although may be due to the low or nule Results and discussion occurrence of these nematodes in soil. According to this, the only description of Adults of the rhabditid nematode males for this species was presented by Ali Diploscapter coronata were observed in Asghar Shah and Shavish Vaid, 2015. two of the four sampled orchards. A total Cats, dogs, and human infections by of nine females were obtained: six from D. coronata were also reported (5, 12). In the agricultural soil for tomato growing this case, the life cycle is characterized by and three from roots. Nematodes were an adult worm, generally inhabiting the characterized by annulated cuticle, two stomach wall. Mature female produces pairs of lips in the mouth region; lips trans- fertilized ova that are excreted with the formed into a pair of medial, outwardly host's feces; eggs are spread by rain into acting, distally bifurcate fossores and a ponds, canals and rivers, where they pair of lateral lamellae; rhabditoidal type of oral cavity and absence of glottoid larvae are then ingested accidentally by a apparatus, vulva median with a crosswise host.hatch Therefore, into first stagepotentially larvae; serious the infective conse- quences could be produced when people presented in table 1 (page XXX). are exposed to contaminated environ- slit (figure 1 A-C, page XXX). Measures are ments with infected feces. A. Salas et al.

Figure 1. Light micrographs of Diploscapter coronata female: A. Anterior end. Note the Vulval region. C. Entire worm. Figura 1. Fotos al microscopio óptico de hembras de Diploscapter coronata. A. twoExtremo fossores anterior. and the pairNótese of lamellae los dos fosores in the head y el parregion. de lamellas B. en la región cefálica.

B. Región vulvar. C. Nematodo vista general.

The clinical symptoms include Worms may establish themselves and epigastric tenderness, diarrhea, crampy multiply in diseased female urino-genital abdominal pain, weakness and nausea (5). systems as well as in hypohydrochloric Studies showed that Diploscapter stomachs, and live as juvenile in the skin, coronata was found in great abundance particularly in cases of scabies. Athari and in all stages of development in human and Mahmoudi (2008), described the stomachs containing little or no hydro- report of a man who developed a mild chloric acid (6, 12). gastrointestinal discomfort including diarrhea associated with feces containing many larvae of Diploscapter coronata. Table 1. Morphometry of Diploscapter coronata females, isolated in present work and those provided by the bibliography. All measurements: mean ± s.d (range) are in µm except V%, (NA: not available). Tabla 1. Morfometría de Diploscapter coronata (hembras) determinada en el presente trabajo y aquellas proporcionadas por la literatura. Todas las medidas: media ± desvío (rango) se expresan en µm excepto V% (N/A: no disponible).

Populations of D. coronata mean ± s.d (range)

Abolafia, J.; Peña- Eyualem et al., 1998 Eyualem et al.,1998 Characters Present work Santiago, 2007 (Ethiopian population) (Iranian population)

Total length 437.6±39.5 (358–504) 427.5±25 (395-480) 350.9±23.4 (317-403) 357.04±75.99 (270-464.3)

Stoma width N/A 3±0.2 (2-3) 2.3±0.1 (2-2.5) 2.32±0.00 (2.32)

Stoma length 18.3±1.4 (16-22) 21.8±0.9 (20-23) 20.9±1.2 (19-23) 19.47±1.24 (18.6-20.8)

Esophagous length 95.3±5.0 (90-109) 108.5±5.2 (95-114) 67.5±2.7 (64-73) 83.96±2.99 (81.2-88.1)

Beginning of basal bulb from anterior end N/A N/A N/A 67.72±2.57 (64.9-69.6)

Nerve ring from anterior end 70.5±4.0 (66-85) 70±3 (65-77) 66.5±3.1 (60-71) 54.28±5.59 (48.7-60.3)

Excretory pore from anterior end 78.0±9.0 (69-100) 79±4.9 (70-89) 75.1±3.9 (67-82) 73.74±8.72 (60.32-83.5)

Maximum body width 26.3±3.1 (20-32) 24.3±1.8 (21-28) 21.7±1.5 (20-25) 24.11±3.86 (18.6-27.8)

Vulva body width N/A N/A N/A 23.88±4.73 (16.2-27.8)

Posterior end width 12.0±1.7 (9-17) 11.3±1 (10-14) 9.6±0.8 (9-12) 19.92±4.22 (16.2-25.5)

Tail length 68.8±5.9 (55-80) 55.5±6.3 (45-71) 51±9 (36-67) 54.26±12.23 (41.7-69.6)

Vagina length 7.3 ± 0.8 (7-9) N/A N/A 6.03±1.27 (4.6-6.9)

V % N/A 53.2±1.5 (50-55.7) 53.7±2.1 (50.1-58.4) 53±4.00 (48-58%) A. Salas et al.

agricultural production models, subsis- the rhabditiform larvae of tence farming, in which much of crop stercoralisNematodes, a soil-transmitted were first diagnosed helminth, as production is used to maintain the farmer, causal agent of , commonly and the farmer’s family, leaving the rest found in tropical and subtropical areas. for sale or trade. In some of them, dogs, Authors considered that while there cats and poultry live together in contact are few reports of these worms as a real with crops, without any health control. parasite, the presence of Diploscapter For these reasons, the isolation of larvae in feces, should be considered in the D. coronata in this study in greenhouses differential diagnosis of Strongyloididasis under this type of agricultural management in endemic areas, because of the highlights the divulgation of hygiene and similarity between larvae of D. coronata, prevention measures in activities involving and Strongyloides stercoralis under soil management, to avoid contamination microscopic examination (5). of fruits and vegetables and infections in D. coronata domestic animals and humans. is also based on their potential as vectors The detection of this nematode in the of food-borneThe health pathogenicsignificance bacteriaof to the surface of fruits and vegetables in contact of the Diploscapter genus with the species with soil (9). D.present coronata work, in Argentina.constitutes the first report In Argentina and other regions of

South-America, is common to find in References

Spain. The Genera (Osche, 1952) Dougherty, 1953 and Diploscapter 1. Abolafia, J.;Cobb, Peña-Santiago, 1913, with R. Description 2007. Nematodes of P. spiculocrestata of the Order Rhabditida sp. n. and froma Species Andalucía Protorhabditis Oriental, Key. Journal of Nematology. 39: 263-274. 2. Agrios, G. N. 2005. Plant pathology. New York. Ed. Elsevier Academic. 922 p. 3. Ali Asghar, S.; Shavish, V. 2015. Description of Sclerorhabditis miniata n. sp. and First Description of Male of Diploscapter coronatus Cobb, 1913 (Nematoda: ). Journal of Nematology. 47(2): 153-158. 4. Andrassy, I. 1983. A taxonomic review of the suborder Rhabditina (Nematoda: ). Paris. ORSTOM. 241 p. 5. Athari, A.; Mahmoudi, M. R. 2008. Diploscapter coronata and review of Literature. Iranian Journal of Parasitology. 3: 42-47. 6. Chandler, A. C. 1938. D. coronata as a facultative parasiteInfection of man, in Iran: with Report a general of the review first case of vertebrate parasitism by rhabditoid worms. In the Journal of Parasitology. 30: 44-55. 7. Cobb, N. A. 1913. New nematode genera found inhabiting fresh water and non-brackish soils. Journal of Washington Academy of Sciences. 3: 432-433. 8. Eyualem, A.; Karegar, A.; Nabil, H.; De Ley, P. 1998. A redescription and ultrastructural study of Diploscapter coronatus (Cobb, 1893) Cobb, 1913 from Ethiopia and Iran. Russian Journal of Nematology. 6(1): 17-22.

Diploscapter sp. strain LKC25, a bacterivorous nematode from soil, as a vector of 9. Gibbs, D. food-borneS.; Anderson, pathogenic G. L.; Beuchat, bacteria L. toR.; preharvest Carta, L. K.; fruits Williams, and vegetables. P. L. 2005. Journal Potential of Applied role of Enviromental Microbiology. 71(5): 2433-2437. 10. Manzanilla-López, R. H.; Marbán-Mendoza, N. 2012. Practical Plant Nematology. México. Ed. Colegio de Post-graduados. 876 p. 11. Martinotti, M. D.; Castellanos, S. J.; González, R.; Camargo, A.; Fanzone, M. 2016. Efecto nematicida de extractos de ajo, orujo de uva y alperujo de aceituna; sobre Meloidogyne incognita, en vid, cv Chardonnay. Revista de la Facultad de Ciencias Agrarias. Universidad Nacional de Cuyo. Mendoza. Argentina. 48(1): 211-224.11. 12. Morimoto, N.; Korenaga, M.; Yagyu, K. 2006. Morphological observations and the effects of Diploscapter coronata from a Japanese patient. Journal of Helminthology. 80: 341-348. 13. Thorne, artificialG. 1961. Principles digestive of fluids Nematology on the survival of

. New York. Ed. McGraw Hill Book Co. 553 p.

Acknowledgements We would like to thank Dr. Eliseo Chaves for his knowledge, cooperation and predisposition during this research.