Molecular and biochemical diversity among isolates of Radopholus spp. from different areas of the World

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Journal o f Nematology 28(4):422-430. 1996. © The Society o f Nematologists 1996.

Molecular and Biochemical Diversity Among Isolates of

Radopholus

spp. from Different Areas of the World

G. A. FALLAS, 1 M. L . HAHN, 2 M. FARGETTE, 1 P. R. BURROWS, 2 J. L. SARAH 1'3 Abstract: Eleven isolates ofRadopholus similis from various banana-growing areas around the world and one isolate o f R. bridgei from turmeric in Indonesia were compared using DNA and isoenzyme analysis. T h e polymerase chain reaction (PCR) was used to amplify a fragment o f ribosomal DNA (rDNA), comprising the two internal transcribed spacers (ITS) and the 5.8S gene. Restriction frag- m e n t length polymorphisms (RFLPs) in this rDNA fragment were used to compare the 10 isolates. T h e analysis o f this rDNA region revealed little variation among the isolates tested. However, data also were obtained by r a n d o m amplified polymorphic DNA (RAPD) analysis o f total DNA, and a hierarchical cluster analysis of these data arranged the R. similis isolates into two clusters. T h e first cluster consisted o f isolates from Nigeria, Cameroon, Queensland, and Costa Rica; the second was comprised o f isolates from Guinea, Guadeloupe, the Ivory Coast, Uganda, and Sri Lanka. T h e isolate o f R. bridgei from turmeric in Indonesia appeared to be more divergent. This grouping was consistent with that obtained when phosphate glucose isomerase (PGI) isoenzyme patterns were used to compare the R. similis isolates. T h e results from both RAPD analysis and PGI isoenzyme studies indicate that two gene pools might exist within the R. similis isolates studied. No correlation could be detected between the genomic diversity as determined by RAPD analysis and either geographic distribution o f the isolates or differences in their pathogenicity. The results support the hypothesis that R. similis isolates have been spread with banana-planting material.

Key words: biochemical systematics, biodiversity, burrowing nematode, isoenzyme, PCR, Radopho- lus similis, RAPD, rDNA, RFLP.

A p p a r e n t biodiversity exists among isolates of the burrowing nematode, Radopho- lussimilis (Cobb, 1893)Thorne, 1949, from

different geographic origins as revealed by differences in pathogenicity (7,12,19) and in vitro reproductive fitness (6). Since the agricultural problems associated with burrowing n e m a t o d e infestations are strongly influenced by the relative pathogenicity and host range of particular nematode isolates, there is a need to assess the diversity o f R. similis as fully as possible in order to

facilitate the development o f integrated control schemes.

Electrophoretic analysis of enzymes has been used for identification and characterization of plant-parasitic nematodes (10). Recently, molecular biological methods, based on the polymerase chain reaction

Received for publication 11 August 1995.

l Laboratoire de Ndmatologie C1RAD FLHOR-- ORSTOM, BP 5035, 34032 Montpellier, Cedex, France.

2 Entomology and Nematology Department, 1ACR- Rothamsted, Harpenden, Hertfordshire, AL5 2JQ, UK.

s Author to whom correspondence may be sent. The au- thors are grateful to the European Economic Community and the Natural Resources Institute for partly supporting this study (Contracts no. TS3*--CT92-0104, and X0196, respectively). We also thank D.T. Kaplan for recommendation of useful oligonucleotides for the RAPD analysis.

E-mail: [email protected].

422

(PCR), also have been applied to the identification and d i f f e r e n t i a t i o n o f plant- parasitic nematodes. Approaches such as the use of random amplified polymorphic DNA (RAPD) analysis (3,14,15,17,23) and the specific amplification of fragments o f the ribosomal DNA (rDNA) cistron (4,8, 13,14,22) have been applied successfully to practical problems in phytonematology.

T h e aim of this study was to assess the diversity between 11 isolates of R. similis

and one isolate ofR. bridgei using biochem-

ical and molecular methods. An attempt was made to correlate geographic origin o f the isolates with apparent genomic or biochemical similarity.

MATERIALS AND METHODS

Nematode Cultures: DNA studies involved

10 Radopholus isolates. Nine o f these were

originally extracted from roots o f banana plants (Musa AAA) in Uganda (Labbubbo,

Kyadondo), Nigeria (Port Harcourt), the Ivory Coast (Anguddddou), Cameroon (Is- land 5, Sanaga River), Guinea (Balikoure), Costa Rica ( T a l a m a n c a ) , G u a d e l o u p e ( N e u f c h ~ t e a u ) , Q u e e n s l a n d (Mac Kay State), and Sri Lanka (Hantane), and have

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Diversity of Radopholus spp.: Fallas et al. 423 been identified by morphology as R. similis

(M. R. Siddiqi, pers. comm.). T h e 10th isolate, collected from turmeric (Curcuma ze- doaria) in East Java, Indonesia, is morpho- logically different from R. similis and has recently been described as a new species, R. bridgei (20). All R. similis isolates were maintained in the laboratory in monoxenic culture at 27°C (+0.5°C) on carrot discs (5,16). For extraction, n e m a t o d e s were rinsed f r o m culture j a r walls aseptically with sterile distilled water and pelleted by c e n t r i f u g a t i o n . N e m a t o d e s were either used immediately for DNA extraction or stored in a minimal volume of sterile distilled water at - 8 0 ° C . T h e isolate of R. tn'idgei was cultured on excised maize roots, a n d n e m a t o d e s were r e c o v e r e d as described previously (11).

For isoenzyme studies, the R. bridgei isolate was not included, and two R. similis isolates collected f r o m b a n a n a roots in Martinique (Morne Rouge) a n d Kenya (Ogembo, Oyugis) were included in addition to the isolates listed above. Nematodes were pelleted by centrifugation and stored in sterile distilled water at -20°C.

DNA extraction: DNA from the 10 nematode isolates was extracted as follows: stan- dard 3qxl pellets of packed nematodes (approximately 4,000 juveniles and adults) were t r a n s f e r r e d to micro-homogenizer tubes (BioMedix: 2 West Avenue, Pinner, "HA5 5BY, U.K.) and 7 I~l of lysis buffer (2) was added per pellet. T h e nematodes were homogenized for 15 seconds followed by centrifugation at 2,000g for 5 seconds. Su- pernatants were transferred to sterile 0.5- ml E p p e n d o r f tubes. For each sample, the pellet was resuspended in 10 p~l lysis buffer and then homogenized and centrifuged as before. T h e supernatants and the pellet were mixed, a n d the c a p p e d tube was placed in a 95°C heating block for 3 minutes. Finally, the samples were centrifuged for 8 minutes at 14,000g, and 17 ~l of su- p e r n a t a n t was collected a n d stored at

- 45oc.

Amplification of the 5.8S/ITS region of ribo- somal DNA and RFLP analysis: T h e frag- m e n t o f the ribosomal DNA (rDNA) com-

prising the two internal transcribed spacers (ITS) and the 5.8S gene was amplified by the polymerase chain reaction (PCR) using primers based on conserved sequences in the 18S and 26S rDNA genes o f Cae- norhabditis elegans (9). T h e p r i m e r sequences were as in Vrain et al (22):

18S primer-

5' T T G A T T A C G T C C C T G C C C T T T 3' 26S primer-

3' G G A A T C A T T G C C G C T C A C T T T 5'. Radopholus DNA (0.2 txl of crude extract- see above) and Globodera rostochiensis DNA, which was used as a positive control, were amplified in 50-~1 reaction volumes con- sisting of 10 mM Tris-HC1, pH 8.8, 50 mM KC1, 15 mM MgC12, 0.1% Triton X-100, 200 ~M dNTPs, 30 pM each primer, and 2 U Taq DNA Polymerase (Stratagene). Am- plification conditions were as follows: 1 cycle of 3 minutes at 94°C, 1 minute at 55°C, 1 minute at 72°C; 38 cycles of 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C; finally, 1 cycle o f 1 m i n u t e at 94°C, 1 minute at 55°C, and 5 minutes at 72°C (13).

Restriction digestions were carried out on 6 to 10 ~1 of the PCR reaction mixture containing the amplified rDNA product using each of six restriction enzymes: H h a I, Hpa II, Rsa I, Hae III, Taq I, and Alu I (Gibco Co., BRL). Reaction conditions were according to supplier's instructions. PCR products were resolved by electro- phoresis through 1% agarose gels in l x TBE buffer. DNA was stained with ethid- ium bromide, visualized on a UV transillu- minator, and photographed with Polaroid Type 667 film. A 123 base pair ladder was used as a size marker.

Random amplified polymorphic DNA analy- sis: For each isolate, PCR reactions were always performed in duplicate using two different volumes (i.e., 0.1 p,1 and 0.2 ~1) of crude DNA extract. Radopholus total DNA was amplified in a final volume of 25 ~xl. Reactions were prepared on ice a n d consisted of 10 mM TrisHCl (pH 8.8), 50 mM KC1, 1.5 mM MgC12, 0.001% gelatin, 20txM of each dNTP, 1 unit of Taq DNA Polymerase (Stratagene), and 20-25 pM of

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424

Journal of Nematology, Volume 28, No. 4, December 1996

TABLE 1. List o f o l i g o n u c l e o t i d e s u s e d as p r i m - ers in t h e R A P D analysis. Primer sequence (5' to 3') Source-Re~rence C A G G C C C T T C O P E R O N A G T C A G C C A C O P E R O N G A A A C G G G T G O P E R O N C A A T C G C C G T O P E R O N C T T C A C C C G A O P E R O N A C G G C G T A T G O P E R O N A A C G G T G A C C O P E R O N G T C T C C G C A A O P E R O N G G G A C G T T G G O P E R O N G G G T G T G T A G O P E R O N K I T l A code OPA-01 K I T A code O P A - 0 3 K I T A code O P A - 0 7 K I T A code OPA-11 K I T E code O P E - 0 9 K I T E code O P E - 1 9 K I T E code O P E - 2 0 K I T K code O P K - 0 2 K I T M code O P M - 1 2 K I T T code O P T - 1 2 I Operon Instruments Alameda, CA.

primer. T e n different primers were used (Table 1), some of them having proved to detect p o l y m o r p h i s m in

Radopholus

spp (15). Amplification was done in a Hybaid O m n i G e n e T h e r m o Cycler. PCR conditions were as follows: 2 minutes at 85°C; 41 cycles o f 1 minute at 92°C, 1 minute at 35°C, 1 minute at 72°C, with the final cycle r e m a i n i n g at 72°C for 7 minutes (11). RAPD products were resolved by electro- phoresis t h r o u g h 1.3% agarose (Sea Kem G T G FMC) gels in 1 x TAE buffer. DNA was stained, visualized, and photographed as above. A 123 base pair ladder was used as a size marker.

Genetic data analysis:

RAPD bands were scored for each primer as present (1) or absent (0), and only DNA bands unambig- uously present in both duplicates were included in the data analysis. Fragments that appeared to be the same molecular size were assumed to represent the same genomic locus due to the mainly intraspecific nature of the present study. T h e simple matching coefficient was used to calculate a similarity matrix. T h e matrix was used to p e r f o r m hierarchical cluster analysis based on the unweighted pair-group method using arithmetic averages (UPGMA) (21). Cluster analysis on all RAPD data was d o n e using 'Genstat' statistical software

(18).

Isoenzyme analysis:

Pellets o f 10,000 n e m a t o d e s (juveniles a n d adults) were g r o u n d twice for 15 seconds in a micro-

homogenizer tube (BioMedix) containing 40 Ixl of 1% glycine. Each sample was centrifuged at 2,000g for 5 seconds between homogenizations. S u p e r n a t a n t was t h e n centrifuged at 14,000g for 5 minutes and 10-~1 samples taken from the top o f the supernatant and placed on the isoelectric focusing mask. Proteins were separated by isoelectric focusing in ultrathin layer (0.15 mm thick) polyacrylamide gels, pH 3-10 ( S e r v a l y t P r e c o t e s , 125 x 125 ram, SERVA: 7 Carl-Benz Str., D-6900 Heidel- berg, Denmark) according to m a n u f a c - turer recommendations. Electrofocusing was carried out at 4°C using a Multiphor II apparatus (Pharmacia Biotech, France). T h e current was adjusted to deliver an ini- tial voltage of 200. Running time for gels was 2 to 3 hours. PGI activity was detected using the staining procedure described in Allendorf et al. (1).

R E S U L T S

Amplification of the rDNA fragment and

RFLP analysis:

T h e amplification of the 5.8 rDNA gene and flanking ITS spacer re- gions of each

Radopholus

isolate yielded one fragment of approximately 0.92 kilobases (Kb) (Fig. 1A, lanes a-j). T h e positive control DNA of

Globodera rostochiensis

(Fig. 1A, lane k) showed a fragment of approximately 1.23 Kb, and the negative control (without template DNA; Fig. 1A, lane 1) gave no amplification products. In gen- eral, digestions of the 0.92Kb rDNA fragment with each of six restriction enzymes revealed little variation between the

R. si-

milis

isolates. Some differences between isolates were observed with two enzymes: Alu I and Hae III. With Alu I additional restriction fragments of 0.55 Kb and 0.70 Kb were present in the isolates from Costa Rica and Uganda (Fig. 1D, lanes h and f, respectively), while after digestion with Hae III an additional restriction fragment of 0.65 Kb was present in the isolates from Guinea and the Ivory Coast (Fig 1E, lanes b and d). No differences in size and num- ber of restriction fragments were obtained with Hha I and Hpa II (data not shown).

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x a b c d e f g h i j k l x II u -0.61 - 0 . 2 5 x a b c d e f g_h i

j mnx

W

Kb

,,,,,0.86

,,,0.61

W

re

x a b c d e f g h i j m n x Kb - 0 . 8 6 - 0 . 4 9 - 0 . 2 5

x a b c d e f g h

i j m n x

_{K b} - 0 . 9 8 - 0 . 6 1

Fro. 1. A) Amplified region of rDNA comprising the 5.8S gene and the flanking internal transcribed

spacers from nine isolates of Radopholus similis and

one of R. bridgei (lanes a-j). a: Nigeria, b: Guinea, c:

Guadeloupe, d: the Ivory Coast, e: Cameroon, f:

Uganda, g: Queensland, h: Costa Rica, i: R. bridgei

(Indonesia), and j: Sri Lanka. Lane k: fragment of

Globodera rostochiensis. Lane 1 : control without nematode DNA. B) Ras I. C) Taq I. D) Alu I. E) Hae III digestion fragments patterns of the rDNA fragment

(lanes a-j: isolates of Radopholus as described above).

Lane m: digestion fragments of lambda Hind III

marker. Lane n: uncut rDNA fragment of Radopho-

lus. Molecular size values given in Kilobases (Kb) were estimated from 123 ladder molecular size marker (lanes x).

T h e isolate o f R. bridgei c o u l d be differ-

e n t i a t e d f r o m t h e b u r r o w i n g n e m a t o d e isolates u s i n g t h r e e o f the six restriction e n z y m e s : Rsa I, T a q I, a n d Alu I (Figs. 1B, 1C, a n d I D ; lane i). Digestion o f the r D N A f r a g m e n t with Rsa I (Fig. 1B) yielded o n e a d d i t i o n a l b a n d o f a p p r o x i m a t e l y 0.20 Kb, while T a q I resulted in s o m e additional re-

striction f r a g m e n t s (Fig. 1C). I n contrast, a f r a g m e n t o f 0.25 Kb g e n e r a t e d by digestion with Alu I was p r e s e n t only in the R.

similis isolates (Fig. 1D, lanes a-h,j).

R A P D analysis: T h e R A P D profiles gen- e r a t e d in this study readily allowed the in- terspecific s e p a r a t i o n o f R . bridgei a n d R.

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426 Journal of Nematology, Volume 28, No. 4, December 1996 all of the R. similis isolates. Three of the 10

RAPD profiles produced are shown in Fig. 2. Primer OPA-07 (Fig. 2A) yielded five main bands from DNA of the R. bridgei isolate (i) that ranged in size from approximately 0.70 Kb to 2.46 Kb. All the isolates o f R. similis (Fig 2A, lanes a-h,j) yielded two main RAPD fragments, one of 1.0 Kb and another of 1.40 Kb. However, only the isolates f r o m Nigeria (a), Cameroon (e), Queensland (g), and Costa Rica (h) yielded an additional band of 2.46 Kb that also appeared to be present in R. bridgei (i). Sep- aration of the same four R. similis isolates was also possible with primers OPA-20 and OPT-12 (Figs. 2B and 2C). Here, these isolates (Nigeria, C a m e r o o n , Q u e e n s l a n d , and Costa Rica) were found to have RAPD bands of 1.30 Kb (Fig. 2B) and 0.98 Kb (Fig. 2C) not observed in the other isolates. Primer OPA-20 (Fig. 2B) yielded a band of 1.54 Kb that was present in all the isolates

°

I

except R. bridgei (i). Interestingly, this primer also generated a f r a g m e n t of approximately 2.1 Kb that was found only in the isolate from Nigeria (Fig. 2B, lane a). T h e i s o l a t e s f r o m N i g e r i a (a) a n d Cameroon (e) revealed two fragments o f 1.54 Kb and 2.21 Kb not f o u n d in any o f the o t h e r isolates. Primer OPT-12 also g e n e r a t e d two fragments, 1.80 Kb a n d 1.90 Kb, from the Guinea, Guadeloupe, Ivory Coast, Uganda, and Sri Lanka isolates (Fig. 2C, lanes b-d, f, j, respectively). T h e hierarchical cluster analysis carried out on the RAPD data from this study was based on a total of 104 scorable RAPD bands generated by the 10 primers. T h e resultant similarity matrix (Fig. 3) was used to compile a d e n d r o g r a m (Fig. 4). T h e analysis separated the R. similis isolates into two groups that were 75% similar to each other. T h e first group contained the isolates from Nigeria, Cameroon, Queens-

y a b c d e f g h i i x ~ x a b c d e f g h i j x Kb

. 2 . 0 9 .1.48 .1.23

x a b c d e f g h i _Kh

Fxo. 2. Random amplified polymorphic DNA profiles (in duplicate) from nine isolates of Radopholus similis and one of R. bridgei. Banding patterns produced by 10-mer primers: A) OPA-07, B) OPA-20, C) OPT-12. Key to isolates as in Fig. 1. Molecular size values given in Kilobases (Kb) were estimated from 123 ladder molecular size marker (lanes x).

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NIG GUI 67.3 - GUA 70.1 97.2 IVC 67.3 98.1 95.3 CAM 95.3 70.1 72.9 UGA 69.2 96.3 99.1 QUE 84.1 81.3 80.4 COR 88.8 78.5 81.3 R. bridgei 37.4 30.8 31.8 SRL 68.2 97.2 94.4

Diversity of Radopholus spp.: Fallas et al. 427

70.1 94.4 72.0 81.3 86.9 79.4 - 78.5 91.6 80.4 95.3 32.7 38.3 32.7 32.7 95.3 71.0 93.5 82.2 37.4 79.4 29.9

NIG GUI GUA IVC CAM UGA QUE COR R. SRL

bridgei

FIG. 3. Genomic similarity coefficient matrix of nine isolates of Radopholus similis and one o f R. bridge'i

obtained f r o m presence a n d absence of a total of t04 RAPD bands generated by the ten 10-met primers used. Matrix is based on the unweighted pair-group method using arithmetic averages (UPGMA). NIG: Nigeria, GUI: Guinea, GUA: Guadeloupe, IVC: Ivory Coast, CAM: Cameroon, UGA: Uganda, QUE: Queensland, COR: Costa Rica, and SRL: Sri Lanka.

land, and Costa Rica (Fig. 4 isolates a, e, g and h, respectively). T h e second group was comprised o f the R. similis isolates

f r o m G u i n e a , G u a d e l o u p e , the I v o r y Coast, Uganda, and Sri Lanka (Fig. 4 isolates b - d , f, and j, respectively). The isolate o f R. bridgei from turmeric in Indonesia

(Fig. 4, isolate i) was only 33% similar to the R. similis isolates, based on these data.

Isoenzyme analysis: Isoenzyme analysis of

PGI was repeated five times, and patterns proved to be stable. As shown in Fig. 5, the

R. similis isolates collected from banana

roots were s e p a r a t e d into two distinct groups with very different PGI profiles. T h e first group, in which the PGI pattern of each m e m b e r consisted of three bands, c o n t a i n e d the isolates f r o m Sri Lanka, Guinea, Guadeloupe, the Ivory Coast, and Uganda (Fig. 5, lanes a, c, e, g, and h). The second group contained the isolates from Nigeria, C a m e r o o n , Q u e e n s l a n d , Costa Rica, and Martinique (Fig. 5, lanes b, d, f,

p, and m), and here the typical isozyme p a t t e r n c o n s i s t e d o f two b a n d s . T h e grouping of the isolates on the basis of PGI was consistent with the two groups differ- entiated by the RAPD analysis.

DISCUSSION

Although the rDNA 5.8S/ITS region has been used successfully to differentiate species and isolates o f phytonematodes, the diagnostic value of this D N A f r a g m e n t varies greatly with the particular nematode species being studied. Sequence diversity in this region, as d e t e r m i n e d by RFLP analysis, has been reported among isolates o f Xiphinema americanum (22) and espe-

cially Aphelenchoides sp. (13), while in Het- erodera glycines this region was f o u n d to be

conserved (8). For R. similis, this particular

region of rDNA might be too conserved to be of diagnostic value without direct D N A sequence comparison. This result confirms

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428 Journal of Nematolog),, Volume 28, No. 4, December 1996 isolates R. btfdgei G U A ~ _ UGA SRL -- GUI - IVC -

ouE

COR NIG C A M I I I I I I I I 100 90 80 70 60 50 40 30 percentage similarity

Flo. 4. Dendrogram showing the relationships between nine isolates of Radopholus similis and one of R. bridgei, generated from genomic similarity coeffi- cients presented in Fig. 3. Codes to isolates as in Fig. 3.

lates f r o m C a m e r o o n a n d N i g e r i a a r e m u c h m o r e p a t h o g e n i c o n b a n a n a s t h a n the isolates f r o m Q u e e n s l a n d o r Marti- n i q u e (7,12).

Based o n the cluster analysis, n o c o r r e - l a t i o n was f o u n d b e t w e e n g e o g r a p h i c p r o x i m i t y o f the original isolates a n d ap- p a r e n t g e n o m i c similarity. F o r e x a m p l e , African isolates are f o u n d in each o f t h e two g r o u p i n g s as d e f i n e d by R A P D a n d PGI analysis. Similar results have b e e n re- p o r t e d with Heterodera schachtii isolates analysed by RAPD (3). H o w e v e r , the rela- tionship b e t w e e n g e o g r a p h i c o r i g i n a n d a p p a r e n t molecular/biochemical similarity o f the Radopholus isolates c o u l d p e r h a p s be u n d e r s t o o d in relation to a s i m u l t a n e o u s s p r e a d o f the n e m a t o d e s t o g e t h e r with ba-

-t-

p r e v i o u s studies c a r r i e d o u t by K a p l a n (14).

T h e level o f g e n o m i c diversity a m o n g b u r r o w i n g n e m a t o d e isolates d e t e c t e d in this w o r k by R A P D analysis is consistent with r e s u l t s o b t a i n e d in r e c e n t s t u d i e s (11,15). H o w e v e r , R A P D d a t a a n d PGI analysis in t h e p r e s e n t s t u d y a r r a n g e d the R. similis isolates into two g r o u p s , probably r e f l e c t i n g d i f f e r e n t g e n e pools. While Ka- plan et al. (15) r e p o r t e d the p r e s e n c e o f a R A P D b a n d associated with citrus parasit- ism in some b u r r o w i n g n e m a t o d e p o p u l a - tions f r o m Florida, it is n o t yet clear if individual isolates o f the g r o u p s established h e r e s h a r e c o m m o n biological o r p a t h o - g e n i c c h a r a c t e r i s t i c s . A c t u a l l y , in b o t h g r o u p s some isolates d i f f e r in their p a t h o - genic characteristics. As an e x a m p l e , the isolates f r o m U g a n d a a n d Sri L a n k a have b e e n s h o w n to vary greatly in t h e i r r e p r o - d u c t i v e fitness a n d p a t h o g e n i c i t y o n ba- n a n a (7,12), yet they w e r e a r r a n g e d in the same g r o u p . I n the s e c o n d g r o u p , the iso-

a b c d e f g h p m i m i J m m m m m m u I i X . m ' n m . - . . m I m m

Fxo. 5. Phosphate glucose isomerase patterns of 10 Radopholus similis isolates collected from banana roots. a: Sri Lanka, b: Nigeria, c: Guinea, d: Cameroon, e: Guadeloupe, f: Queensland, g: the Ivory Coast, h: Uganda, p: Costa Rica, m: Martinique.

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Diversity of Radopholus spp.: FaUas et al. 429

nana-planting material. It is possible that

R. similis was introduced into the Carib- bean from West Africa (J. Bridge, pers. c o m m . ) . I n o u r s t u d y , isolates f r o m Cameroon and Nigeria are present in the same group, and although the isolate from Martinique was not included in the RAPD analysis, the PGI patterns of these three isolates w e r e identical. This indicates strongly their possible close relatedness. In addition, based on both RAPD and PGI analysis, the two isolates from Guinea and the Ivory Coast appear to be similar. In- terestingly, b a n a n a p r o d u c t i o n in the Ivory Coast developed mainly in the late 1950s, when growers from Guinea left their country after independence and set- tled in the Ivory Coast, introducing their own planting material into the area. The similarity between the Guinea and Ivory Coast isolates is also revealed in the rDNA, as the enzyme Hae III generates an additional restriction fragment only in these two isolates (Fig. 1E). In contrast, the his- tory o f banana production in Cameroon, which has a common b o r d e r with Nigeria, is completely separated from Guinea and the Ivory Coast; this is reflected in the g r o u p i n g of the R. similis isolates from these countries. Detailed studies of the his- torical spread o f banana plants may help our understanding o f the geographic distribution o f R. similis isolates.

T h e use o f molecular and biochemical tools e m p l o y e d in this study, combined with host range and pathogenicity assays, should provide us with information essen- tial for more successful control of burrowing nematodes.

LITERATURE CITED

1. Allendorf, F. W., N. Mitchell, N. Ryman, and G. Stahl. 1977. Isoenzyme loci in brown trout (Salmo

trutta L.): Detection and interpretation from popula-

tion data. Hereditas 86:179-190.

2. Black, W. C., N. M. Duteau, G.J. Puterka, J. R. Nechols, and J. M. Pettorini. 1992. Use of the random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) to detect DNA polymorphisms in aphids (Homoptera: Aphididae). Bulletin of Ento- mological Research 82:151-159.

3. Caswell-Chen, E. P., V. M. Williamson, and F. F. Wu. 1992. R a n d o m amplified polymorphic DNA

analysis of Heterodera cruciferae and H. schachtii populations. Journal of Nematology 24:343-351.

4. De Giorgi, C., S. M. Finetti, M. Di Vito, and F. Lamberti. 1992. A fragment of the large subunit of rDNA gene amplified by polymerase chain reaction in individual nematodes. Nematologia Mediterranea 20:149-152.

5. Fallas, G., and J. L. Sarah. 1994. Effect of stor- age temperature on the in vitro reproduction of Ra-

dopholus simili~. Nematropica 24:175-177.

6. Fallas, G., and J. L. Sarah. 1995. Effect of temperature on in vitro multiplication of seven Radopho-

lus similis isolates from different banana-producing

zones of the world. Fundamental and Applied Nema- tology 18:445-449.

7. Fallas, G., J. L. Sarah, and M. Fargette. 1995. Reproductive fitness and pathogenicity of eight isolates of Radopholus similis on banana (cv. Poyo). Nematropica 25:135-141.

8. Ferris V. G., J. M. Ferris, and J. Faghihi. 1993. Variation in spacer ribosomal D N A in some cyst- forming species of plant-parasitic nematodes. Funda- mental and Applied Nematology 16:177-184.

9. Files, J. G., and D. Hirsh. 1981. Ribosomal DNA

of Caenorhabditis elegans. Journal of Molecular Biology

149:223-240.

10. Fox, P. C., and H . J . Atkinson. 1986. Recent developments in the biochemical taxonomy of plant- parasitic nematodes. Agricultural Zoological Reviews

1:301-331.

11. Hahn, M. L., P. R. Burrows, N. C. Gnanapra- gasam, J. Bridge, N. J. Vines, and D. J. Wright. 1994. Molecular diversity amongst Radopholus similis populations from Sri Lanka detected by RAPD analysis. Fundamental and Applied Nematology 17:275-281. 12. Hahn, M. L., J. L. Sarah, M. Boisseau, N . J . Vines, J. Bridge, D.J. Wright, and P. R. Burrows. 1996. Reproductive fitness and pathogenic potential of selected Radopholus populations on two banana cul- tivars. Plant Pathology 45:223-231.

13. Ibrahim, S. K., R. N. Perry, P. R. Burrows, and D.J. Hooper. 1994. Differentiation of species and populations of Aphelenchoides and of Ditylenchus angus- tus using a fragment of ribosomal DNA. Journal of Nematology 26:412-421.

14. Kaplan, D. T. 1994. Molecular characterization of the burrowing nematode sibling species, Radopho-

lu~ citrophilus and R. similis. Pp. 77--83 in F. Lamberti,

C. De Giorgi, and D. M. Bird, eds. Advances in molecular plant nematology. Plenum Press: New York.

15. Kaplan, D. T., M. C. Vanderspool, C. Garrett, S. Chang, and C. H. Opperman. 1996. Molecular polymorphism associated with host range in the highly conserved genomes of burrowing nematodes,

Radopholus spp. Molecular Plant Microbe Interactions

9:32-38.

16. O'Bannon, J. H., and A. L. Taylor. 1968. Mi- gratory endoparasitic nematodes reared on carrot discs. Phytopathology 58:385.

17. Pastrik, K.J., H . J . Rumpenhorst, and W. Burgermeister. 1995. Random amplified polymorphic DNA analysis of a Globodera paUida population

(9)

4 3 0 Journal of Nematology, Volume 28, No. 4, December 1996 selected for virulence. Fundamental and Applied

Nematology 18:109-114.

18. Payne, R. W., P. W. Lane, A. E. Ainsley, K. E. Bicknell, P. G. N. Digby, S. A. Harding, P. K., Leech, H. R. Simpson, A. D. Todd, P. J. Verrier, R. J. White, J. C. Gower, G. Tunnicliffe Wilson, and L.J. Patter- son. 1987. Genstat 5 reference manual. Oxford, UK: Clarendon Press.

19. Sarah, J. L., C. Sabatini, and M. Boisseau. 1993. Differences in pathogenicity to banana (Musa sp., cv. Poyo) among isolates of Radopholus similis from different production areas of the world. Nematropica 23:75-79.

20. Siddiqi, M. R., and M. L. Hahn. 1995. Radopho- lus bridgei sp.n. (Tylenchida: Pratylenchidae) from In-

donesia and its differentiation by morphological and molecular characters. Afro-Asian Journal of Nema- tology 5 : 3 8 4 3 .

21. Sneath, P. H. A., and R. R. Sokal. 1973. Nu- merical taxonomy. San Francisco, CA: Freeman.

22. V r a i n , T. C., D. A. W a k a r c h u k , A. C. Levesque, and R. I. Hamilton. 1992. Intraspecific rDNA restriction fragment length polymorphism in the Xiphinema americanum group. Fundamental and Applied Nematology 15:563-573.

23. Williaras, J. G. K., A. R. Kubelik, K.J. Livak, J. A. Rafalski, and S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research 18:6531- 6535.