First report of two Mesocriconema ( Nematoda : Criconematidae ) species in mulberry trees in Costa Rica 1

Mulberry is a native species from temperate zones of Asia, which has spread worldwide (Benavides et al. 1994). In Central America, particularly in Costa Rica, it is propagated by cutting plants directly. Its foliage is used as fodder for cattle and goats (Rodríguez et al. 1994, González 1996) and it can reach 12 meters high, being adapted as a fast growing shrub, with vigorous leaf shoots (Ting-Zing et al. 1988). It also presents an excellent nutritional value, due to its high levels of protein (20-24%) and digestibility (75-85%) (Benavides 1995). Costa Rica is endowed with a suitable climate for the cultivation of different crops, but there are constraints affecting its agriculture, such as diseases and plant pests, mainly nematodes. The Criconematidae family is a taxonomy commonly known as ring nematodes, due to the ABSTRACT RESUMO

Soil samples were collected from the rhizosphere of mulberry (Morus alba L.) trees, in a 1,000 m 2 plot, at the Experimental Farm Santa Lucia, in Barva, Heredia, Costa Rica, in August 2013. The plants showed symptoms of yellowing, declining and poor development. Specimens of two ring nematode were collected from the soil and identified as Mesocriconema sphaerocephalum ) Loof 1989and M. anastomoides (Maqbool & Shahina 1985 Loof & De Grisse 1989, based on the morphological and morphometrical analysis of females. Both nematodes have been previously found in Costa Rica. However, this is the first report of nematodes from the Criconematidae family associated to mulberry trees and it provides additional information on the distribution of this phytoparasite. KEY-WORDS: Mesocriconema sphaerocephalum  vegetation (Tarjan 1971, López-Chaves 1980, López-Chaves & Azofeifa-Chacón 1980, López-Chaves & Salazar-Figueroa 1987a, Arroyo et al. 2004, Wingching-Jones et al. 2008, Powers et al. 2011, Guzmán-Hernández et al. 2011). This study aimed to report the presence of ring nematodes (Mesocriconema sphaerocephalum and M. anastomoides) in mulberry trees, in Costa Rica.
The isolation and identification of nematodes were performed in the Laboratory of Nematology of the Universidad Nacional de Costa Rica, in Heredia, Costa Rica. Nine soil samples, which were placed in sealed plastic bags and stored at 4ºC, before analysis, were collected from a 1,000 m 2 plot, at the Experimental Farm Santa Lucia, in Barva (10º01'34.40"N, 84º06'49.60"W, altitude of 1,220 m, average rainfall of 2,693 mm per year and average temperature of 23ºC), Heredia, which belongs to the School of Agricultural Sciences of the Universidad Nacional de Costa Rica, in August 2013. The area had been previously planted with sorghum, which had showed symptoms of yellowing and poor development.
Nematodes were extracted from soil samples by flotation-centrifugation (Jenkins 1964), killed and fixed in hot 4% formaldehyde (70ºC). Subsequently, fifteen females from each species were infiltrated with glycerin, by using the Seinhorst's modified slow method (Seinhorst 1959 and1962), and mounted on slides, for observation and preservation. All specimens were deposited in the Criconematid Collection, in the Laboratory of Nematology of the Universidad Nacional de Costa Rica.
Both species were morphologically and morphometrically identified by using data collected by Geraert (2010). All measurements were taken in micrometers (μm) and in the format mean ± S.D. (standard deviation range), using an Olympus BX50 microscope and a stage micrometer. Images of key morphological features were taken with a Nikon DS-Fi1 camera and edited with the aid of Photoshop CS4. The classification of nematodes was based on Siddiqi (2000).
Five years after planting, mulberry trees showed symptoms of yellowing, declining and poor development. These general symptoms are often difficult to distinguish, because they are associated to other root pathogens and soil fungi. So, it is important to make a nematode analysis to detect phytoparasites, as proved in this investigation.

anastomoides (Maqbool & Shahina 1985) Loof & De Grisse 1989
Mesocriconema species presents a cuticle with retrorse annuli, mean annular tickness of 4.7 μm, smooth uneven or crenated margins and no posterior appendages. Lateral field is often present, usually by anastomoses. The first head annulus is seldom separated and the second head annulus is usually wider than the first. Submedian lobes are present, rarely absent. Labial plates are present or absent. Open vulva and anterior vulva lip are ornamented in several species. The post-vulval body is usually short, with round or truncate terminus, seldom acute. Males have a lateral field with 4 lines. The bursa is usually well developed, with enclosing terminus, rarely narrow and short. The tail terminus is round to acute. Juveniles present smooth uneven or crenate annuli posterior margins, never with appendages, and with annuli in profile never drawn out posteriad (Geraert 2010).
The genus Mesocriconema is closely related to Criconemoides, but it differs by having an open vulva and a difference in the structure of the submedian lobes (Loof & De Grisse 1989). It is not always easy to evaluate these differences and both genera are synonymized by Hunt et al. (2005). On the other hand, Geraert (2010) uses the genus Mesocriconema as proposed by Brzeski et al. (1998).
M. sphaerocephalum has a cuticular retrorse annuli with irregular margins. It's a small nematode, ventrally arcuate and characterized by the presence of a high number of anastomoses throughout the body, forming a zig-zag pattern. The anterior body end appears truncated, labial disc is low, submedian lobes and labial plates are small and not protruding. Stylet is robust (about 51.0 µm), knobs 8.0-12.0 present a concave or anchor shape (about 10 µm). Criconematid oesophagus is typical. The vulva is open, simple, with rounded lips, devoid of projections. Gonad is often recurved, sometimes even twice. The post-vulval part is rounded. The vagina is straight and the postvulval body part is short, with a rounded tail. Males were not found. All the principal morphometric values of the specimens are in agreement with the ranges described by Geraert (2010). The population of M. sphaerocephalum reported here has a similar morphometrics to the description made by Cordero et al. (2012).
The species can be compared to M. microdorum and M. sphaerocephala.