RESEARCH/INVESTIGACIÓN MOLECULAR IDENTIFICATION, RACE DETECTION, AND LIFE CYCLE OF ROTYLENCHULUS RENIFORMIS IN EGYPT M. Adam1*, S. F. Diab1, A. Farahat1, A. A. Alsayed1, and H. Heuer2 1Department of Zoology and Nematology, Faculty of Agriculture, Cairo University, Egypt; 2Julius Kühn- Institut, Federal Research Centre for Cultivated Plants, Institute for Epidemiology and Pathogen Diagnostics, Braunschweig, Germany; *Corresponding author: [email protected] ABSTRACT Adam, A., S. F. Diab, A. Farahat, A. A. Alsayed, and H. Heuer. 2018. Molecular identification, race detection, and life cycle of Rotylenchulus reniformis in Egypt. Nematropica 48:59-67. Molecular and bioassay methods were used to characterize an isolate of Rotylenchulus reniformis collected from Fowa-Kafr-El-Sheihk, Egypt, and maintained for 40 years under greenhouse conditions. The identification of this isolate was rapidly determined by PCR with species-specific primers developed for amplifying the D2–D3 of 28S rRNA gene. The nested PCR allowed direct sequencing without cloning the fragments. All sequences of the D2–D3 of 28S rRNA fragments obtained were identical and exhibited 99% similarity with a partial 28S ribosomal RNA gene of R. reniformis in GenBank. Phylogenetic analysis revealed relationships of our isolate with R. reniformis from other countries and a sister relationship with R. macrosoma. The isolate’s reproductive potential was tested using different hosts. The isolate infected and reproduced on cowpea, castor, and cotton with differences among the plants. Cowpea was the most preferred host. Low reproduction was achieved on cotton. After penetration, the minimum time for the appearance of mature females was 3 days on cowpea, 7 days on castor, and 14 days on cotton. Complete formation of egg masses was observed after 7, 14, and 21 days on cowpea, castor, and cotton, respectively. Juvenile females of the second generation were recorded after 14 days on cowpea and 21 days on castor and cotton. Key words: D2–D3 of 28S rRNA gene, Egypt, life cycle, molecular diagnosis, pathogenicity, Rotylenchulus reniformis RESUMEN Adam, A., S. F. Diab, A. Farahat, A. A. Alsayed, and H. Heuer. 2018. Identificación molecular, detección de razas y ciclo de vida de Rotylenchulus reniformis en Egipto. Nematropica 48:59-67. Se usaron métodos moleculares y de bioensayo para caracterizar un aislado de Rotylenchulus reniformis recogido de Fowa-Kafr-El-Sheihk, Egipto, y se mantuvo durante 40 años en condiciones de invernadero. La identificación de este aislado se determinó rápidamente por PCR con cebadores específicos de especie desarrollados para amplificar el gen D2-D3 del rRNA 28S. La PCR anidada permitió la secuenciación directa sin clonar los fragmentos. Todas las secuencias de D2-D3 de los fragmentos de rRNA 28S fueron idénticas y exhibieron un 99% de similitud con un gen de ARN ribosómico 28S parcial de R. reniformis en GenBank. El análisis filogenético reveló las relaciones de nuestro aislado con R. reniformis de otros países, y tuvo una relación hermana con R. macrosoma. El potencial reproductivo de los aislamientos se probó usando diferentes hospederos. El aislamiento se infectó y reprodujo en caupí, ricino y algodón con diferencias entre las plantas. El caupí fue el anfitrión más preferible. Se logró una baja reproducción en algodón. Después de la penetración, 3 días fueron el tiempo mínimo para la aparición de hembras maduras en el caupí, 7 días en el ricino y 14 días en el algodón. La formación completa de masas de huevos se observó después de 7, 14 y 21 días en caupí, ricino y algodón, respectivamente. Se registraron hembras juveniles de la segunda generación después de 14 días con caupí y 21 días con ricino y algodón. 59 60 NEMATROPICA Vol. 48, No. 1, 2018 Palabras claves: ciclo de vida, D2-D3 del gen 28S rRNA, diagnóstico molecular, Egipto, patogenicidad, Rotylenchulus reniformis INTRODUCTION documented for a single population of R. reniformis (Koenning et al., 2000; Robbins et al., The reniform nematode, Rotylenchulus 2001; Robbins et al., 2002; Usery et al., 2005). reniformis, is one of the most common plant- Additionally, multiple populations of R. parasitic nematode species in tropical and reniformis document variation in reproduction and subtropical regions (Robinson et al., 1997). This pathogenicity across a variety of crops (McGawley nematode is an obligate sedentary semi- et al., 2010). Observations by Birchfield and endoparasite on the roots of plants and is Brister (1962) led them to postulate the existence considered a major pathogen of cotton and other of races or pathotypes of R. reniformis. The first crops in Egypt and several other countries definition of R. reniformis races was reported by (Ibrahim et al., 2010). Successful nematode Dasgupta and Seshadri (1971a), who designated management requires accurate information on the race A and race B based on differential species and pathogenicity of a given nematode reproduction patterns on cotton, castor, or cowpea. population causing the crop damage. Most Vadhera and Shukla (1999) identified another Rotylenchulus species can be identified using population of R. reniformis that failed to reproduce distinctive morphological characters of immature on castor and considered it as another race. Rao females (Robinson et al., 1997). However, high and Ganguly (1996) reported the existence of four intraspecific variability of some diagnostic physiological races of the reniform nematode that features makes identification to species level of differed in their ability to reproduce on cotton, this group based on morphology very difficult. castor, cowpea, bajra, and mustard. McGawley et Currently, DNA-based approaches have al. (2010) found, among 6 isolates of R. become the preferred means for identification of reniformis, that there were differences in their Rotylenchulus species as these methods are faster reproduction and pathogenicity on cotton and and more accurate than morphology (Agudelo et soybean. al., 2005; Leach et al., 2012; Nyaku et al., 2013; In the Nile Delta region of Egypt, 13 Van Den Berg et al., 2016). During the past few populations of Rotylenchulus spp. were collected decades, several studies have been conducted to in nematological surveys undertaken during the characterize nuclear ribosomal RNA (rRNA) 1970s on several crops, including cotton, sugar genes of R. reniformis (Subbotin et al., 2006; Zhan beet, wheat, rice, soybean, and ornamental plants et al., 2011; Palomares-Rius et al., 2017). such as jasmine. These populations were divided Conventional and real-time PCR with species- into two different pathotypes based on their specific primers were developed for diagnostics of physiological reaction on 13 soybean cultivars R. reniformis using sequence differences between (Farahat, 1979). Of them, only one population that species in rRNA and β-tubulin genes (Showmaker showed highest reproduction rate was able to et al., 2011; Sayler et al., 2012). The 18S internal survive so far under greenhouse conditions. This transcribed spacer (ITS) and the D2 and D3 population was recovered from severely damaged expansion segments of 28S rRNA genes have been roots of jasmine plants grown in fields located at shown to be markers for the molecular diagnostics Kafr-El-Sheikh Governorate, one of the most and diversity of species of Rotylenchulus important agricultural areas for cotton cultivation. (Subbotin et al., 2007; Palomares-Rius et al., Therefore, the objective of this research was to 2017). Based on differences in the D2-D3 of 28S characterize a population of Rotylenchulus spp. rRNA gene sequences, PCR with species-specific maintained on pigeon pea (Cajanus cajan) under primers was developed for rapid diagnostics of R. greenhouse conditions since 1977 i) molecularly reniformis (Van Den Berg et al., 2016). and ii) biologically. The life cycle of R. reniformis is host type, host susceptibility, temperature, and nematode MATERIALS AND METHODS population dependent. The life cycle of R. reniformis ranged from 14 to 29 d on different Nematode source hosts (Lim and Castillo, 1978; Rodriguez, 1978; Vadhera et al., 2001; Rashid and Khan, 2013). The reniform nematode used in this study was Rotylenchulus reniformis has been documented to obtained from a pure culture reared on pigeon pea be quite variable in pathogenicity (McGawley and (C. cajan) under greenhouse conditions at the Overstreet, 1995; Nakasono, 2004; Arias et al., Zoology and Agricultural Nematology 2009). Marked variation in reproduction among Department, Faculty of Agriculture, Cairo different cultivars of a single host species has been University. The initial inoculum was taken from Identification, race, and life cycle of R. reniformis in Eqypt: Adam et al. 61 naturally infected jasmine plants (Jasminum template in a second PCR with identical amounts grandiflorum) collected in 1977 from Fowa region of the reaction mixture mentioned above. The in Kafr-El-Sheikh Governorate, Egypt. The pure nested PCR products were purified by using the culture was established from a single egg mass, QIAquick PCR purification kit (Qiagen, Clinilab, and morphological characters of the corresponding Egypt). PCR products were analyzed on an ABI female suggested similarity to R. reniformis 373 automated DNA sequencer and sequenced in (Robinson et al., 1997). For culture propagation, both directions using the same primers. The single egg-masses picked from the females were obtained sequences were compared with individually inoculated onto pigeon pea plants