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Journal of Nematology 44(4):391–398. 2012.� The Society of Nematologists 2012.
Observations on the Foliar Nematode, Aphelenchoides besseyi,Infecting Tuberose and Rice in India
MATIYAR R. KHAN,1 ZAFAR A. HANDOO,2 UMA RAO,3 S. B. RAO,4 J. S. PRASAD5
Abstract: The foliar nematode Aphelenchoides besseyi causes white tip disease in rice (Oryza sativa L.) and floral malady in tuberose(Polianthes tuberosa L.). This nematode is widely distributed in the rice fields of many states of India, including West Bengal (WB),Andhra Pradesh (AP), Madhya Pradesh (MP) and Gujarat (GT). In order to generate information on intraspecific variations ofA. besseyi as well as to confirm the identity of the nematode species infecting these important crops, morphological observation wasundertaken of A. besseyi isolated from tuberose and rice from WB and rice from AP, MP and GT. The molecular study was only donefor rice and tuberose populations from AP and WB. The variations were observed among the populations in the tail, esophageal andanterior regions, including the occurrence of four as well as six lateral lines in the lateral fields. The morphometrics of observedpopulations showed variations and those could be regarded as a consequence of host-induced or geographical variations. PCRamplification of the rDNA ITS 1 and 2 region of rice (AP) and tuberose (WB) populations of A. besseyi generated one fragment ofapproximately 830 bp, and the size of the ITS region was 788 bp and 791 bp for tuberose and rice population, respectively. Alignmentof the two sequences showed almost 100% similarity. Blast analysis revealed a very high level of similarity of both the Indian strains toa Russian population. The Indian and Russian strains could be differentiated using restriction enzyme Bccl. Host tests revealed thatrice (cv. IET 4094), oat (cv. OS-6) and teosinte (cv. TL-1) showed a typical distortion due to the infection of A. besseyi. Five germplasmlines of oat showed no infection of the nematode under field conditions. Local cultivars of onion, maize, chrysanthemum, gladiolus,and Sorghum halepense were also not infected by A. besseyi.
Key words: Aphelenchoides besseyi, diagnosis, distribution, host, India, morphology, rice, tuberose.
Amongst nematodes associated with rice (Oryza sativa L.)cultivation in West Bengal (WB), white tip nematode,Aphelenchoides besseyi is one of the most important dueto its quarantine significance. Dastur (1936) reportedits occurrence for the first time in rice from the formerCentral Provinces, now Chattisgarh region of MadhyaPradesh (MP), and later the nematode was reported wide-spread in India, particularly in the rice growing areasof Uttar Pradesh, MP, Tamil Nadu (Muniappan andSeshadri, 1964; Sivakumar, 1987), Gujarat (GT), AndhraPradesh (AP) (Savitri et al., 1998) and Tripura (Nathet al., 1995). A serious outbreak of white tip diseasewas observed in 60% of the rice cultivars, with the mostseriously affected cultivars being H.R.12 and Pankaj inAP ( Jayaprakash and Joshi, 1979). The nematode causesyield losses of 10-15% in rice in GT (Thakar et al., 1987).The white tip disease of rice is severe in the southernand eastern states, where yield losses of 20% mayoccur (Prasad et al., 1986).
On tuberose (Polianthes tuberosa L.), the foliar nem-atode was first discovered to induce foliar disease inHawaii (Holtzmann, 1968). Later, the occurrence of‘floral malady’ caused by A. besseyi in tuberose wasfound in the Ranaghat areas of the Nadia district of
WB (Chakraborti and Ghosh, 1993). Subsequently,A. besseyi was proven to be a pest of rice and tuberose inWB (Khan, 2001). The species was reported to causea serious foliar disease of tuberose in the Mekong Deltaof Vietnam, and based on morphological criteria andribosomal RNA gene sequencing, the nematode wasidentified to be A. besseyi (Cuc and Pilon, 2007). Wide-spread occurrence of the white tip nematode infectingrice was reported from WB (Das and Khan, 2007, Khanand Das, 2009).
The widespread detection of white tip disease to ricein the southern and eastern states of India was reportedand the foliar nematode problem is restricted to tube-rose in WB and Odisha. Earlier studies were mostly onthe occurrence of the problems and a detailed morpho-logical and molecular evaluation of this nematode wasthought to be essential for a better understanding ofthe species. Our preliminary light microscopic studies onthe nematodes from these populations showed con-siderable variations in the body and stylet length in-cluding in the shape of tail, tail terminus and numberof lateral lines. The objectives of this study were toevaluate populations of A. besseyi obtained from tube-rose in WB and from rice in AP, MP, GT, and WB, usinglight and scanning electron microscopy (SEM) observa-tions and molecular analysis and to assess the diagnosticvalue of morphological and molecular characters amongthe populations and to confirm the species infectingboth rice and tuberose in India. Detailed morphometricand molecular observations of the nematode popula-tions on rice and tuberose are presented herein.
MATERIALS AND METHODS
Five populations of A. besseyi collected from rice(Oryza sativa L.) grains from Hyderabad, Andhra Pradesh
Received for publication August 9, 2012.1Department of Agricultural Entomology, Bidhan Chandra Krishi Viswavidyalaya,
Nadia 741235, West Bengal, India.2USDA-ARS, Nematology Laboratory, Beltsville, Maryland 20705, USA.3Division of Nematology, Indian Agricultural Research Institute, New Delhi-
110 012, India.4The Ella Foundation, Hyderabad, India.5Directorate of Rice Research (ICAR), Hyderabad, India.The authors are grateful to Dr. D. J. Chitwood for suggestions and review of
the manuscript and to David Martel and Joseph Mowery for excellent technicalassistance.
Mention of trade names or commercial products in this publication is solelyfor the purpose of providing specific information and does not imply recom-mendation or endorsement by the United States Department of Agriculture.
E-mail: [email protected] paper was edited by Andreas Westphal.
391
(AP), Jabalpur, Madhya Pradesh (MP), Gujarat (GT),and West Bengal (WB) where A. besseyi populations in-fect both rice and tuberose, were studied for morpho-metric variations. Nematodes were extracted from ricegrains after pounding with a mortar and pestle. Thecrushed grains were placed on single-ply tissues fittedover wire screens, which were then placed in Petridishes containing tap water. The water level in the Petridish was maintained to touch the bottom of the screenand tissue paper so that the nematodes could easilymove down to the water below. Another Petri dishwas placed on top of the screen and tissue paper to retardthe evaporation of water. The entire assembly was keptfor 15 to 20 hours at room temperature (30 to 358C).Aphelenchoides besseyi from fresh, infected tuberose(Polianthes tuberosa L.) flower stalks obtained from foliarnematode-infested fields were extracted following themethod of Khan and Pal (2001). Nematode specimenswere killed in a hot-water bath, fixed in 3% formaldehydeand subsequently processed by the Seinhorst method(Seinhorst, 1959). The processed specimens were thenmounted in anhydrous glycerine on glass slides. Photo-micrographs of males, females and juveniles were takenwith a Color Digital Camera (Retiga EXi, Q-Imaging,Austin, TX) attached to a compound microscope (aLeica Wild MPS48 Leitz DMRB, Leica Microsystems,Wetzlar, Germany). Altogether 24 parameters from atleast 30 female of A. besseyi from each location and host inIndia were studied, as well as 30 males from WB ontuberose. The de-Man ratios and other values were de-termined from measurements taken with the help ofan ocular micrometer (Hooper, 1986). For SEM, livingspecimens were fixed in 3% glutaraldehyde buffered with0.05 M phosphate (pH 6.8), dehydrated in a graded seriesof ethanol, critical-point dried from liquid CO2, andsputter coated with a 20 to 30-nm layer of gold-palladium.
Collection of nematode populations for DNA extraction:DNA was extracted from the populations of A. besseyiinfesting tuberose in WB and rice in AP. Tuberose flowerstalks showing distortions caused by foliar nematodeswere cut and placed in sterile spring water in a sterilePetri plate (5 cm diameter). After about an hour, nem-atodes that had migrated to the water were transferredinto 1.5 ml microcentrifuge tubes, pelleted and used forDNA extraction. Similarly, rice seeds collected fromnematode-infested rice fields were placed in water anddissected to allow the nematodes to move into the water.
Extraction of nematode DNA: DNA was extracted fromthe above two populations separately according to themethod described by Subbotin et al., (2000). Ten tofifteen ml of sterile distilled water was added into eachtube containing nematodes, which were crushed witha microhomogenizer. About 50 ml of nematode lysisbuffer (125 mM KCl, 25 mM Tris-HCl pH 8.3, 3.75 mMMgCl2, 2.5 DTT, 1.125 % Tween 20, 0.025% gelatin)and 2 ml of proteinase K (600 g/ml) were added. Thetubes were incubated at 65 8C (1h) and 95 8C (10 min)
consecutively. Tubes were centrifuged at 16,000 g for1 min and the supernatant containing DNA was storedat -20 8C until further use.
PCR amplification and cloning: Five ml of the DNAsuspension was added to the PCR reaction mixturecontaining 0.2 mM dNTP9s, 0.2 mm of each primer(reverse and forward) and 2.5 ml 10 x Taq incubationbuffer (10 mM Tris-HCl pH 8.3, 50 mM KCl, 1.5 mMMgCl2), 200 mM of each dNTP, 1 unit of Taq polymerase(Bangalore Genei, India), and double distilled water toa final volume of 25 ml. Primers (59) TTGATTACGTCCCTGCCCTTT (39) and (59) TTCACTCGCCGTTACTAAGG (39) as described by Joyce et al. (1994) foramplification of ITS 1 and 2 were used in the PCR re-action. Amplification reactions were performed in anEppendorf Gradient thermal cycler. Each cycle haddenaturation at 95 8C for 30 seconds, annealing at 55 8Cfor 30 seconds, and extension at 72 8C for 90 seconds; thecycle was repeated 44 times with a final extension of 72 8Cfor 5 min. Five-ml aliquots of amplification productswere loaded on 1.2 % agarose gels prepared in 1X TAE(Tris-acetate-EDTA) buffer (pH 8.0), separated by electro-phoresis (5 V/cm for 2.5 h) and stained with ethidiumbromide. The gels were visualized using a gel documen-tation system (Vilber Lourmat, France). PCR productswere purified with a PCR purification kit (Qiagen,Germany) as per manufacturer’s protocols, and 2 mlof purified product was used for TA cloning using pGEMT vector (Promega, Madison, USA). Recombinant colo-nies were identified using an ampicillin and the bluewhite colony selection method according to the manu-facturer’s instructions, and were confirmed by PCR usingthe same primers used for PCR amplification. Plasmidswere prepared and the inserts were sequenced andanalyzed. Multiple sequence alignment was done byMUSCLE, alignment curation by Gblock, phylogeneticanalysis by PhyML and tree construction by TreeDyne(Dereeper et al., 2008).
Host range tests: Onion (Allium cepa L. cv. Bombai red),chrysanthemum (Chrysanthemum sp.), gladiolus (Gladiolussp. cv. Souvik), five germplasm of oat (Avena sativa L. cv.0S-387, JO-03-309, JHO-2010-4, OL-1690, UPO-10-3),maize (Zea mays L. cv. Rajkumar), Sorghum halepense (L.)Pers.-Gramma-1, rice (Oryza sativa L. cv. IET 4094) andteosinte (Euchlaena mexicana Schrad. cv. TL-1) were grownin foliar nematode-infested plots at the Central ResearchFarm, Bidhan Chandra Krishi Viswavidyalaya, Gayespur,WB. The onion, oat, maize, Sorghum halepense, gladiolusand rice were grown in a plot of 10 m x 1 m insertedbetween the heavily infested plots of tuberose infectedby A. besseyi. The crops were grown during 2009-2011 ina standard spacing of the crops during their growingseasons. The teosinte was sown in rows in a plot of5 m 3 2.5 m with spacing of 40 cm between plants and60 cm between rows. The teosinte plants (one of thethree rows) were inoculated with tuberose foliar nem-atodes extracted from tuberose at 30, 45 and 60 days
392 Journal of Nematology, Volume 44, No. 4, December 2012
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Aphelenchoides besseyi in India: Khan et al. 393
FIG. 1. Photomicrographs of Aphelenchoides besseyi female. A. Anterior end with esophagus; B-C: mid body region; E-F: Tail end; G-H-I: Tail tip,A-I: 20 mm scalebar.
394 Journal of Nematology, Volume 44, No. 4, December 2012
after sowing at 500 nematodes/plant. After flowering, theplants were examined for infection by A. besseyi both infield and laboratory. Ten chrysanthemum plants wereraised for two consecutive years (2009-2010) in pots andinoculated artificially by dispensing a nematode suspen-sion of A.besseyi with a 5 ml dropper on the foliage andterminal points of 30 day-old cuttings at 2000 nema-todes/plant. Infection by A. besseyi and development ofsymptoms on the plants were monitored regularly duringthe growing periods of the crops in both years. Five plantsamples from ten sites within an experimental plot foreach crop were collected and analysed in the laboratoryfor detection and estimation of nematode population.The observations on nematode population multiplica-tion on the above crops were recorded from grains (100grains) as well as from whole plants (at least five of onionand gladiolus) or plant parts (ten leaves including thegrowing points of chrysanthemum).
RESULTS
Morphometrics and Morphology: Morphometrics ofA. besseyi females and males are given in Table 1. Thenematode body length varied from 514 mm to 845 mm
in females. The body length indicated that it wasmoderately to highly variable in all the studied A. besseyipopulations, whereas the ratios a, b9, b, c, and c9 werehighly variable in the tuberose population. Stylet length(10-11mm), head to median bulb distance (HMB 53-61mm), and V% (71-79) were moderately to least vari-able. The mean length of the post-uterine sac (PUS) inA. besseyi populations from WB- rice and Gujarat-ricewas relatively larger (40mm) than that of other pop-ulations (34-35mm) in this study. The mean b-ratio forMP-rice, WB-rice and GT-rice was less (9) than that ofWB-tuberose (12) and AP-rice (11).
The A. besseyi populations studied in this investigationshowed variations in the tail shape and terminal mucroand 2-3 processes, esophageal (shape of median bulb)and head end in the anterior regions, including theoccurrence of four as well as six lateral lines in the lateralfields (Fig. 1-2).
PCR amplification of the rDNA ITS 1 and 2 region ofeach of the two studied populations of A. besseyi gener-ated one fragment of approximately 830 bp. However,removal of primer sequences in the flanking 18S and 28Sgave 788 and 791 bp ITS 1 and 2 sequences for rice andtuberose strains, respectively (Fig. 3). Further there was
FIG. 2. SEM photomicrographs of Aphelenchoides besseyi female. A. Head end; B-C: Lateral fields; D: Tail end.
Aphelenchoides besseyi in India: Khan et al. 395
a thymine (T) instead of a guanine (G) at position 169 incase of the rice strain. Sequences of both populationshave been deposited in GenBank under the accessionnumbers JF826519 (rice population) and JF826517(tuberose population). The Indian populations arehighly similar to a Russian population of A. besseyi(GenBank EU186069.1 790 bp). Multiple sequencealignment by Clustalw (Larkin et al., 2007) indicateda gap at position 176 in the sequence of Russian as incase of the tuberose strain. Interestingly, the thymine (T)at position 498 was replaced by cytosine (C) in theRussian population compared to both the sequencesof the Indian strains. This sequence variation gave arestriction site for the enzyme Bccl only in the Russianpopulation that could be used for differentiating itfrom the two Indian strains. The phylogenetic tree in-dicated that the two Indian populations and one Russianof A. besseyi were similar and distant from one populationof A. ritzamabosi (Fig. 4).
Studies on crop hosts of A. besseyi (Table 2) revealedthat the rice (cv. IET 4094), oat (cv. OS-6) and teosinteshowed a typical distortion due to the infection ofA. besseyi. Although, five germplasm lines of oat procured
from the All India Coordinated Research Project onForage Crops (Kalyani Centre, West Bengal) showed noinfection of the nematode under field conditions. Noinfection of A. besseyi on local cultivars of onion, maize,chrysanthemum, gladiolus and S. halepense were recordedeven on the few plants that showed some minor distortionlike symptoms.
DISCUSSION
In this study, morphometrics of A. besseyi populationsfrom AP-rice, MP-rice, WB-rice and WB-tuberose, GT-rice conform with the measurements from the type hostand locality (Christie, 1942), rice population from India(Dastur, 1936), Senegal (Fortuner, 1970), Stylosantheshamata Taubert population (cultured on Alternariaalternata) from Queensland of Australia (Gokte et al.,1992), onion population from Sri Lanka (Lamberti et al.,1996) and rice population from Egypt (Amin, 2002).There were considerable variations in measurementsamong the populations from India, Senegal and Sri Lankadue to genetic variation and or host-induced variability.B’Chir (1977) observed that the population reared onImpatiens balsamina was longer than the ones reared onAlternaria citri. Rajan and Mathur (1990) proposed twogroups of A. besseyi from India and Philippines isolatesbased on morphometry and culturability on fungal hostsand considered Cuttack (eastern India) and Pune(western India) populations belonged to one and Hy-derabad (southern India) and Philippines isolates(Philippines) to another group. However, in the present
FIG. 3. Comparison of ITS 1 and 2 sequences of Aphelenchoides besseyi from India and Russia. Tuberose strain (T): rice strain (R) and Russianstrain (Russian).
FIG. 4. Phylogentic tree among the three populations of Aphe-lenchoides besseyi and Aphelenchoides ritzemabosi.
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study, no appreciable variation in morphometry of riceand tuberose populations of A. besseyi from India wasnoticed. The morphology of A. besseyi populations (riceand tuberose) showed limited variation in the tail shape,medial bulb of esophagus and head end of anteriorregions. Furthermore, the occurrence of four as well assix lateral lines in the lateral fields of the rice pop-ulation was observed in SEM as well as light microscope(LM) observations. Viewing the number of lateral lineswith LM can be difficult in a nematode genus like Ap-helenchoides. Hooper and Ibrahim (1994) also describedA. nechaleos, a very closely related species of A. besseyi andviewed four lines with LM and six lines with SEM.Aphelenchoides nechaleos was also differentiated from14 species of Aphelenchoides but the number of laterallines was not considered as differentiating character.
Host tests indicated that the A. besseyi populationfrom tuberose induces symptoms in oat (cv. OS-6), rice(cv. IET-4094), and teosinte (cv. TL-1) but not onion,maize, S. halepense, gladiolus and chrysanthemum.Lamberti et al., (1996) found A. besseyi inducing a dis-tortion and twisting of stems and discoloration of theleaf apical portion on onion in Sri Lanka, and provedthe casual relationship of the nematode population onstem tissues and disease symptoms and bulb yield loss.Over 67 different crops including maize, chrysanthe-mum and oat are attacked by A. besseyi, among themrice, strawberry and tuberose are the principal hosts inIndia (Khan, 2010). The A. besseyi population fromtuberose was proven earlier to be the same populationcausing white tip disease of rice (Khan, 2001). However,in our study, maize, chrysanthemum and gladiolus werenot infected, probably because of differential prefer-ence of the nematode population for specific hosts.
No genetic difference was found between the rice andtuberose strains of A. besseyi. Absence of the restric-tion site for Bccl in the ITS sequence of Indian strainscould be used to differentiate the Indian strains andthe Russian strains. Cuc and Pilon (2007) identifiedA. besseyi infection in tuberose from the Mekong Deltaof Vietnam using morphological characters and partialsequences of two rRNA genes (small subunit 18S andlarge sub unit 28S). Their BLAST search of the Gen-Bank database also showed homologous sequences of
the 18S rRNA sequence with those of Aphelenchoidesritzemabosi (GenBank DQ901554) and Aphelenchoidesbesseyi isolate 98 from North Florida, USA (GenBankAY508035), with 602/628 (95%) and 595/643 (92%)nucleotide identities in homologous regions, respec-tively. Therefore, the present work provides both mor-phological and molecular sequence variations found inpopulations of A. besseyi infecting rice and tuberose thatwill enable researchers to identify the species infectingboth rice and tuberose and to select DNA-cutting en-zymes for use in molecular identifications tests that willbe more rapid and accurate than conventional methodsbased on LM study. In addition, this study provides fun-damental information that is needed for morphologicaland molecular diagnosis of this species to provide con-tinued protection for the rice and tuberose industry inIndia and throughout the world.
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TABLE 2. Host tests with an Aphelenchoides besseyi population from tuberose.
Plant Cultivar SymptomsZA. besseyi
population/100grains A. besseyi population/5 plants and/or 10 leaves
Onion (Allium cepa L.) Local - 0Maize (Zea mays L.) Local - 0Oat (Avena sativa L.) OS-6 + 760.33 (6-10)Teosinte (Euchlaena mexicana Schrad.) TL-1 + 1060.24 (8-14)Sorghum halepense (L.) Pers. Local - 0Gladiolus sp. Souvik - 0Chrysanthemum sp. Local - 0Rice (Oryza sativa L.) IET-4094 + 1760.32 (16-23)
Z +/- = symptoms appeared/not appeared.
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