Further evidence of the association of a ... - Wiley Online Library

Plant Pathology (2001) 50, 628±636

Further evidence of the association of a phytoplasma and a virus with yellow leaf syndrome in sugarcane S. M. Aljanabi*², Y. Parmessur, Y. Moutia, S. Saumtally and A. Dookun Mauritius Sugar Industry Research Institute, ReÂduit, Mauritius

Using the polymerase chain reaction (PCR), reverse-transcriptase±PCR (RT±PCR) and double-antibody-sandwich enzyme-linked immunosorbent assay (DAS±ELISA), a phytoplasma (sugarcane yellows phytoplasma, ScYP) and a virus (Sugarcane yellow leaf virus, ScYLV) were detected in sugarcane with yellow leaf syndrome (YLS) in Mauritius. Samples were collected from clones undergoing quarantine, in a variety-collection plot and in commercial fields. A 1´25 kb DNA fragment encoding the phytoplasma 16S rRNA was consistently amplified by nested PCR. Of 134 samples with and without symptoms derived from 113 varieties, 111 were infected by either ScYLV or ScYP. The phytoplasma was detected in 63 samples by PCR. Restriction fragment-length polymorphism (RFLP) analysis of the phytoplasma 16S rDNA amplified product indicated that sugarcane yellows phytoplasma group III, which is related to Western X phytoplasma, is present in Mauritius. ScYLV was detected by RT±PCR and ELISA. The virus was more widely distributed than the phytoplasma, and was found in 70 and 100 samples by ELISA and RT±PCR, respectively. There was a significant correlation between the presence of the phytoplasma and YLS symptoms, while such correlation was not significant for ScYLV detected by RT±PCR. ELISA was less sensitive than RT±PCR for detection of ScYLV. Forty-one samples were coinfected with both microorganisms. Eighty-five per cent of the samples displayed symptoms when ScYLV and SCYP coexisted, while 55 and 38% were observed when ScYP or ScYLV, respectively, was present alone. The results indicate that the presence of both organisms enhanced the syndrome. Keywords: ELISA, PCR, Polerovirus, quarantine, RT±PCR, Sugarcane yellow leaf virus, sugarcane yellows phytoplasma

Introduction Yellow leaf syndrome (YLS) of sugarcane was first described in 1989 in Hawaii (Schenk, 1990) and is characterized by yellowing of the midrib. The colour gradually extends to the leaf blade, and is sometimes accompanied by a shortening of the upper internodes, producing a fan-leaf appearance. YLS is prevalent in adult canes, and stress conditions such as cold and moisture deficit tend to favour development of symptoms. It is suspected that symptoms are similar to those of yellow wilt reported in the late 1960s from a number of countries in Central and East Africa (Ricaud, 1968; Rogers, 1970). No cause was found at that time. Following the occurrence of YLS in Hawaii, several countries recorded the syndrome, and it has since been observed in 34 countries (Lockhart & CronjeÂ, 2000). A number of factors, such as abiotic stresses and insect *To whom correspondence should be addressed. ²E-mail: [email protected] Accepted 5 April 2001.

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damage, can produce symptoms similar to those of YLS, rendering identification based on symptoms difficult. YLS has been found to be transmitted by vegetative propagation of cane sets (Schenk & Hu, 1991), suggesting that a pathogen is involved. Subsequently, Lockhart et al. (1996) detected a Luteovirus, named Sugarcane yellow leaf virus (ScYLV), and Vega et al. (1997) also confirmed the presence of the virus. Proof of pathogenicity through transmission of the virus by aphids has been obtained (Scagliusi & Lockhart, 2000). Moonan et al. (2000) classified the virus as a Polerovirus in the family Luteoviridae, based on its biological properties and comparison of its genomic sequence with those of other members of the Luteoviridae. However, the etiology of the syndrome has not been fully resolved. Investigations carried out by CronjeÂ et al. (1996); CronjeÂ et al. (1998) in South Africa showed that a phytoplasma was associated with YLS symptoms. This was based on PCR amplification of the 16S rDNA of the organism and electron microscopy. The phytoplasma was also detected on symptomless plants. It has provisionally been named sugarcane yellows phytoplasma, Q2001 BSPP

Yellow leaf syndrome in sugarcane

and the strains divided into subgroups I and II (ScYP I and ScYP II) based on RFLP profile (CronjeÂ & Bailey, 1999). In sugarcane, four other phytoplasma diseases are known: sugarcane white leaf (ScWL, Nakashima et al., 1994; Wongkaew et al., 1997); sugarcane grassy shoot (ScGS, Wongkaew et al., 1997); sugarcane green grassy shoot (Sdoodee et al., 1999); and Ramu stunt (CronjeÂ et al., 1999). Analysis of the RFLP patterns of PCR products of ScYP 16S rRNA and sequence data analysis of the intergenic spacer region between the 16S and 23S rDNA genes indicated that ScYP I and ScYP II belong to the Western X and ScWL phytoplasma groups, respectively (CronjeÂ et al., 1999). In Mauritius, YLS was first observed in 1994 on variety CP 72 1210. Symptoms were subsequently observed in several other varieties, and the presence of ScYLV was confirmed in 1996 (Saumtally & Moutia, 1997). This study was undertaken to investigate the distribution of YLS in Mauritius; to determine the predominance of the two types of pathogens that have been associated with the disease; and to relate symptoms to the presence of either or both pathogens. Varieties undergoing quarantine were also screened for the pathogens to identify possible routes of entry.

Materials and methods Plant material One hundred and thirty-four leaf samples, derived from 113 varieties, were collected between July and November 1999. The varieties were grown in either a closed quarantine glasshouse, a variety collection plot, or commercial fields island-wide. Material taken from each stool was visually assessed by three scorers for the presence (yellow midrib and yellowing of the lamina) or absence (green leaves) of symptoms. For each variety, one stalk from a cane stool was sampled. Leaves were collected from plants with or without YLS symptoms. A list of varieties tested for ScYLV and ScYP is given in Table 1.

Total nucleic acids extraction Total nucleic acids were extracted following the method of Harrison et al. (1994). Leaf tissue (5 g) was cut into 2 mm strips and ground to a fine powder in liquid nitrogen. The extraction buffer (2% CTAB, 1´4 m NaCl, 20 mm EDTA, pH 8´0, 100 mm Tris-HCl, 0´2% 2mercaptoethanol) was prewarmed to 658C before adding to the ground leaf samples. Four millilitres of extraction buffer per g leaf tissue were added, mixed well and incubated at 658C in a water bath for 60 min. The tubes were then cooled to 258C and an equal volume of chloroform : isoamyl alcohol (24 : 1) was added, mixed well and centrifuged at 4000 g for 10 min. The upper layer was recovered, and an equal Q 2001 BSPP


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Table 1 Detection of Sugarcane yellow leaf virus and sugarcane yellows phytoplasma by PCR, RT±PCR and ELISA in 134 samples derived from 113 varieties Sample No. Varietya

Presence of DAS±ELISAb RT±PCRb PCRc symptoms

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

± 1 ± ± ± ± ± ± 1 ± ± ± ± 1 ± ± 1 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 1 1 ± ± ± ± ± ± ± 1 1 ± 1 ± 1 1 ± ± 1 1 ± ± 1 1

CC 8226 CC 8596 CC 8527 CC 8227 CC 8215 CC 8592 R 832089 R 830288 R 831592 R 830395 R 840075 R 841289 R 832065 R 830547 R 830680 R 840653 R 832276 G 8537 G 75368 G 8758 G 8737 N 27 ROC 15 Q 136 Q 159 Q 154 Q 135 Q 151 Q 138 Q 155 Q127 MEX 70485 SP 803280 SP 80185 ROC 14 ROC 13 MEX 801410 MEX 68134 MEX 79431 MEX 73206 SP 803390 SP 792233 SP 801520 S 17 CO 527 CO 976 CO 1208 CP 62258 CP 65357 CP 67412 CP 70321 CP 72355 CP 72370 CP 79318 CP 681026 CP 681067 CO 975 CP 701527

1 1 1 1 1 ± ± 1 1 1 1 1 1 1 ± 1 1 1 1 ± ± 1 1 ± 1 ± ± ± ± 1 1 ± 1 1 1 1 1 ± ± ± 1 1 1 1 1 1 1 ± 1 1 ± ± 1 1 ± ± 1 ±

1 1 1 1 ± 1 ± 1 1 ± ± 1 1 1 ± ± 1 ± ± ± ± ± ± ± 1 ± 1 ± ± ± ± ± ± 1 1 1 ± ± ± ± 1 1 1 1 ± 1 ± ± ± 1 1 ± 1 ± ± 1 ± 1

1 1 1 1 1 ± ± ± 1 ± ± 1 1 1 ± ± 1 ± ± 1 1 1 ± ± 1 1 1 ± 1 ± ± ± ± ± 1 ± ± 1 ± 1 1 1 1 1 1 ± 1 1 1 1 1 ± 1 1 1 ± ± 1

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S. M. Aljanabi et al.

Table 1. continued

Table 1. continued

Sample No. Varietya


Sample No. Varietya


59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118

1 ± 1 ± 1 1 1 1 1 ± 1 1 1 1 1 1 1 1 1 1 ± 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ± ± ± 1 1 ± 1 ± 1 1 1 1 1 1 ± 1 1 1 ± ± ± 1 1 1

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134

± 1 1 1 1 ± ± 1 ± ± ± ± ± 1 1 ±

CP 721210 F 149 F 166 F 170 F 172 H 328560 H 605657 H 699092 M 13/56 M 555/60 M 574/62 M 527/68 M 587/70 M 744/70 R 570 M 2597/79 M 554/79 M 2077/78 M 2343/77 M 1197/77 M 1176/77 M 937/77 M 134/75 M 1722/71 M 1030/71 M 1682/70 SP 713501 NCO 310 M 1342/84 M 1300/84 M 5/83 M 1412/32 M 784/82 M 175/82 M 36/82 M 33/82 M 3/82 M 1551/80 RP 8068 ROC 5 ROC 2 ROC 1 PB 739067 R 575 Q 96 Q 72 CP 742005 RB 705051 R570 M 1658/78 M 3035/66 M 695/69 R570 M 1557/70 M 1551/80 M 1658/78 M 52/78 M 555/60 R570 R570

1 1 1 1 1 1 1 ± 1 1 1 1 1 1 1 ± 1 ± 1 1 ± 1 1 1 1 1 1 1 ± 1 1 1 1 1 1 1 1 ± ± 1 1 ± ± 1 1 1 1 1 1 1 ± 1 1 1 1 1 ± 1 1 1

1 1 1 ± ± ± 1 1 1 1 1 ± ± ± 1 ± ± ± ± 1 1 ± ± 1 1 ± ± 1 ± ± 1 1 ± 1 1 1 ± ± 1 1 1 1 ± 1 ± 1 1 ± ± 1 1 ± ± 1 ± 1 1 ± ± 1

1 1 1 ± ± 1 1 1 ± 1 1 1 1 ± 1 1 1 1 1 1 ± 1 1 1 1 ± ± 1 ± ± 1 1 ± 1 1 1 1 1 1 1 1 1 ± 1 ± 1 1 1 1 1 ± ± 1 1 ± 1 ± ± 1 ±

R570 M 1658/78 M 1557/70 M 1658/78 M 1658/78 M 3035/66 M 52/78 M 695/69 R570 R570 R573 1658/78 M 96/82 M 695/69 R570 M 695/69

1 1 1 1 1 1 ± 1 1 ± ± 1 1 1 1 1

1 ± ± ± 1 ± ± 1 ± ± ± ± ± ± 1 1

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

a Source of material: samples 1±43 (quarantine); 44±106 (variety collection plot); 107±134 (commercial fields, island-wide). b RT±PCR and DAS±ELISA detect Sugarcane yellow leaf virus. c PCR detects sugarcane yellows phytoplasma. 1, detection positive; ±, not detected.

volume of ice-cold isopropanol was added to each sample. The tubes were mixed and kept at 2208C for 2 h. Nucleic acids were spooled off or precipitated at 3000 g for 10 min. After air-drying the nucleic acid was resuspended in sterile, double-distilled water and used for both phytoplasma and ScYLV detection. DNA quality and quantity were checked by agarose gel electrophoresis.

PCR for diagnosis of phytoplasma Total genomic DNA was subjected to PCR assay using universal primers designed to amplify a specific sequence within the 16S rDNA region of phytoplasmas. Several primer sets were tested, but the nested approach described by Gunderson & Lee (1996), with different first-round primers and thermal cycling profiles, was adopted as it produced consistent results. Primers P6 (5 0 -CGG TAG GGA TCA CTT GTT ACG ACT TA-3 0 , Deng & Hiruki, 1991) and SN910601 (5 0 -CGA AAA AAC CTT ACC AGG TCT TTG-3 0 , Namba et al., 1993) were used for the first-round amplification of the 16S rDNA. For second-round amplification, to amplify an internal fragment of the 16S rDNA of ScYP, primers R16F2n (5 0 -GAA ACG ACT GCT AAG ACT GG-3 0 , Gunderson & Lee, 1996) and R16R2 (5 0 -TGA CGG GCG GTG TGT ACA AAC CCC G-3 0 , Lee et al., 1993) were used. The PCR was performed in a total volume of 25 m L consisting of 1 unit Taq polymerase (Boehringer Mannheim, Germany), 1 £ reaction buffer, 100 m m dNTPs, 0´5 m m of each primer and 50 ng of stock Q 2001 BSPP



DNA for the first round amplification. For the secondround amplification, 1 m L of the PCR product from the first round was used as template DNA. The PCR profile was one cycle at 948C for 3 min, 35 cycles at 948C for 30 s, 538C for 1 min for the first-round amplification and 568C for the second-round PCR, 728C for 1 min 30 s, and finally one cycle at 728C for 10 min. The PCR products were electrophoresed on 1´4% agarose gel, stained with ethidium bromide, and photographed under UV light.

RFLP analysis of PCR products The 16S rDNA products of positive samples amplified by PCR using the nested primers R16F2n and R16R2 were analysed after restriction endonuclease digestion. Five microlitres of each PCR product were digested separately with two restriction enzymes, RsaI and HaeIII. RsaI was used to distinguish between the different groups of phytoplasmas, while HaeIII allows differentiation of phytoplasmas from other prokaryotes. The PCR products were incubated at 378C for 2 h with the restriction enzymes. The digested products were separated by electrophoresis on 2% Metaphore agarose gel (FMC Bioproducts, ME, USA), stained with ethidium bromide, and visualized using UV light.

RT±PCR for diagnosis of ScYLV ScYLV was detected by reverse-transcriptase±polymerase chain reaction (RT±PCR) on total nucleic acids extracted from the youngest fully unrolled leaves. Amplification was carried out using primers YLS 462 and YLS 111, designed on sequence data obtained from the luteovirus group specific fragment cloned from a Florida ScYLV strain (M. Irey, unpublished results) following the protocol described by Comstock et al. (1998). Total nucleic acids (1 m L) and 1 m L primer YLS 462 (15 m m) were boiled for 5 min, quenched on ice and reverse-transcribed at 428C for 15 min, followed by heating at 998C for 5 min to denature the reverse transcriptase. The reverse-transcription solution (10 m L) consisted of 1 £ PCR buffer (Boehringer Mannheim) supplemented with MgCl2 (6´5 mm), dNTPs (1 mm), 10 U RNase inhibitor (Boehringer Mannheim), and 25 U MuLV reverse transcriptase. The cDNA was amplified by PCR in a reaction mixture consisting of the following: 10 m L reverse-transcribed solution, 4 m L PCR buffer (Boehringer Mannheim), 4 m L MgCl2 (25 mm), 1 m L primer YLS 111 (15 m m), 2´5 U Taq polymerase (Boehringer Mannheim) and 30´5 m L water. The thermal profile was: 948C 1 min, 548C 1 min, 728C 20 min, 1 cycle; 948C 1 min, 548C 1 min, 728C 2 min, 40 cycles, and a final elongation at 728C for 10 min. PCR products were electrophoresed on 1´8% agarose gel, stained with ethidium bromide and photographed under UV light. Q 2001 BSPP


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DAS±ELISA Diagnosis of ScYLV by double-antibody-sandwich enzyme-linked immunoassay (DAS±ELISA) was carried out as follows. All leaves from a given stalk were sampled. They were cut into 1´0 £ 0´5 cm2 strips and thoroughly mixed. A subsample of 5 g was ground to a fine powder in liquid nitrogen using a coffee grinder (Moulinex Type 685). A subsample of 0´25 g was mixed with 1 mL 100 mm phosphate buffer (100 mm Na2HPO4, 100 mm NaH2PO4´2H2O pH 6´0) containing 1% Na2SO3 and 0´05% Tween 20. The mixture was allowed to stand for 1 h at 258C with occasional vortexing, then centrifuged at 8000 g for 15 min, and the supernatant used in the assay. Microtitre plates were coated (100 m L per well) with an anti-ScYLV polyclonal antiserum (1 mg mL21) diluted 1000£ in coating buffer (15 mm Na2CO3, 34 mm NaHCO3 pH 9´6). Plates were incubated for 2 h at 378C or overnight at 48C. The plates were washed three times with PBST (137 mm NaCl, 1´5 mm KH2PO4, 8´1 mm Na2HPO4´7H2O, 0´05% Tween 20 pH 7´4) with 3 min soaking in between each wash. After draining, the plates were either used immediately or stored at 2208C. Two replicates per extract (100 m L) were loaded and incubated for 2 h at 378C or at 48C overnight. The plates were washed with PBST and dried. To each well, 100 m L of the anti-ScYLV alkaline phosphatase-conjugated antibody diluted 1 : 500 in TBST (20 mm Tris± HCl, 150 mm NaCl, 0´05% Tween 20 containing 5% nonfat dry milk (Bio-Rad Laboratories, Hercules, CA, USA) were added. Plates were incubated, rinsed and dried as described above. To each well, 100 m L of substrate (p-nitrophenyl phosphate at 0´6 mg mL21 in diethanolamine buffer pH 9´8, containing 1 mm MgCl2) were added and incubated at 378C for 2±4 h or overnight at 48C. The absorbance was read at 405 nm in a Dynatech MR 5000 plate reader. A sample was considered positive if its absorbance value was at least twice that of the healthy control.

Results Detection of phytoplasma by PCR Of the 134 samples representing 113 varieties tested, the expected 1250 bp fragment of the phytoplasma 16S rDNA was amplified from 63 samples in 54 varieties after the second round of amplification, using the nested primers R16F2n and R16R2 (Fig. 1). The first round of amplification using the universal primers P6 and SN910601 also produced a specific band of 1500 bp, but results were not consistent. Forty-seven of the 76 samples with typical YLS symptoms, and 16 of 58 symptomless ones, were positive for ScYP (Table 2). A higher number of samples (41) presented typical YLS symptoms when both the phytoplasma and the virus coexisted (Table 2). Out of the 134 samples tested, only six were positive for the phytoplasma and expressed the

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Figure 1 Polymerase chain reaction (PCR) amplification of 16S rDNA sequence using nested primers R16F2n and R16R2. Lane M, 100 bp molecular weight marker; lanes 2±44, DNA extract from 43 sugarcane varieties in quarantine; lane 45, DNA from variety S17; lane 46, phytoplasma DNA; lane 47, DNA from variety CP 72 1210; lane 48, negative control (no DNA). Arrows indicate the 1´25 kb amplified fragment of the phytoplasma 16S rDNA.

YLS symptoms. There was a positive significant correlation (r 0´36) between the presence of the phytoplasma (including coinfection with the virus) and YLS symptoms in the samples tested. Nineteen out of 43 exotic clones introduced in quarantine were infected with ScYP. ScYLV was detected in 59 of 76 samples showing YLS symptoms (including coinfection with the phytoplasma) and in 41 (71%) of 58 symptomless varieties. Only 18 samples with YLS symptoms were positive for ScYLV.

Restriction digestion of the PCR product with RsaI and HaeIII Digestion of the amplified products with HaeIII followed by electrophoresis confirmed the presence of a phytoplasma in the samples tested. Three fragments of size 110, 180 and 1060 bp were common in all samples Table 2 Summary results of survey for yellow leaf syndrome

Samples

Sy1 Sy1 Sy1 Sy1 Sy± Sy± Sy± Sy± V1 V1 V± V± V1 V1 V± V± Total Ph1 Ph± Ph1 Ph± Ph1 Ph± Ph1 Ph±

Quarantine Variety collection plot Commercial fields Total

43 14 63 24 28 3 134 41

2 13 3 18

1 5 0 6

6 5 0 11

3 2 6 11

10 9 11 30

1 2 2 5

6 3 3 12

Samples were tested for sugarcane yellows phytoplasma (ScYP) and Sugarcane yellow leaf virus (ScYLV). Sy1, sugarcane leaves with YLS symptoms; Sy±, sugarcane leaves without symptoms; V1 or V±, Sugarcane yellow leaf virus detected or failed to be detected by RT± PCR, Ph1 or Ph±, sugarcane yellows phytoplasma detected or failed to be detected by PCR.

digested with HaeIII (Fig. 2). Other prokaryotes, if present, would have given more frequent cuts and numerous small bands. The PCR products digested with RsaI and electrophoresed on 2% agarose gel gave only one pattern consisting of four bands of 110, 270, 340 and 500 bp (Fig. 3).

Detection of ScYLV Seventy samples were found positive for ScYLV by ELISA, and of these 46 had YLS symptoms and 24 did not. Using this method, there was a significant correlation (r 0´23) between the presence of the ScYLV (including coinfection by the phytoplasma) and the YLS symptoms. RT±PCR amplification for ScYLV RNA showed that 100 of the 134 samples were infected with the virus, and the expected fragment size of 352 bp (Fig. 4) was amplified with primers YLS 462 and YLS 111. ScYLV was detected in 59 of 76 samples showing YLS symptoms (including coinfection with the phytoplasma), and in 41 of 58 symptomless varieties. Only 18 samples with YLS symptoms were positive for ScYLV (Table 2). All positive plants detected by ELISA were also found to be positive by RT±PCR, except for five samples (Table 1). For the varieties coming from quarantine, 29 out of 43 tested positive for ScYLV by RT±PCR while only seven were positive by ELISA. Altogether 87 varieties were found positive for ScYLV by ELISA and RT±PCR. The association of symptoms of YLS with the presence of ScYLV using RT±PCR was not significant (r 0´09).

Discussion Yellow leaf syndrome has been associated with ScYLV Q 2001 BSPP



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Figure 2 RFLP analysis of PCR products generated with nested primers combination (R16F2n and R16R2) of the phytoplasma 16S rDNA sequence amplified from positive plants (lane 1±19) using HaeIII restriction enzyme. Lane 20, DNA from variety S17; lanes 21±22, phytoplasma DNA; lane 23, positive plant DNA; lane 24, DNA of variety CP 72 1210; M, 100 bp molecular weight marker. Arrows indicate the restriction fragment size of HaeIII enzyme.

in a number of countries. This is probably because research has been focused on the virus with the availability of a polyclonal antiserum. Scagliusi & Lockhart (2000) presented evidence to support the theory that a virus is the causal agent of one form of the syndrome. They also succeeded in transmitting the virus after inoculation of aphids fed on YLS-infected source plants, and reproducing the symptoms. However, another causal agent was not excluded. Comstock et al. (1998) found that most positive clones containing the virus did not have symptoms. However, with the development of universal primers for the phytoplasma 16S RNA, an increasing number of countries are detecting the phytoplasma in sugarcane. Phytoplasmas,

previously known as mycoplasma-like organisms (MLOs), are associated with diseases in over 300 plant species. On infection with phytoplasma, the host's thylakoid membranes become extremely reduced and disintegrate, and the levels of photosynthetic pigments (chlorophyll A and B) and carotenoids decrease (Plavsic et al., 1988). In virus-infected plants with yellows diseases, the enzyme chlorophylase is activated causing the destruction of chlorophyll (Goodman et al., 1967), and a similar mechanism might be triggered in phytoplasma-infected plants. In Mauritius, yellow leaf syndrome was found to be widespread in several sugarcane varieties and in 30 of 43 clones undergoing quarantine. Despite the high

Figure 3 RFLP analysis of PCR products generated with nested primers combination (R16F2n and R16R2) of the phytoplasma 16S rDNA sequence amplified from positive plants (lanes 1±17) using RsaI restriction enzyme. Lane 18, uncut PCR product of the 16S rDNA. M, 100 bp molecular weight marker. Arrows indicate the size of the restriction fragment produced by RsaI restriction enzyme.

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Figure 4 RT±PCR amplification of a ScYLV sequence using YLS 462 and YLS 111 primers showing the 352 bp fragment (black arrow) of the ScYLV. Samples 2±18 were total nucleic acids extracts of sugarcane varieties from quarantine with and without YLS symptoms. M, molecular weight marker IX (Boehringer Mannheim).

number of ScYLV-positive samples observed, the correlation coefficient with the syndrome was not significant. This is probably due to the high sensitivity of the RT±PCR assay, which can detect low titres of the virus in latently infected canes. RT±PCR detected the presence of ScYLV in 41 (71%) symptomless varieties, and this could explain the nonsignificant correlation between the virus and the symptoms. The use of DAS± ELISA was found to have limitations as it lacked sensitivity. The significant correlation observed between the presence of ScYLV and YLS using ELISA rather than RT±PCR could be explained by the fact that a large number of samples (.30) with no symptoms (Sy±, V1) were found positive for the virus by RT±PCR (Table 2). Surveys in South Africa (CronjeÂ & Bailey, 1999) and Cuba (Arocha et al., 1999) have shown that the phytoplasma is more prevalent in sugarcane with YLS. Two strains of phytoplasmas related to Western-X phytoplasma and sugarcane white leaf (ScWL) phytoplasma, as described by Lee et al. (1998), have been detected in the samples tested in South Africa. The strain detected in Mauritius belongs to Western-X phytoplasma, which is classified as group III according to the classification of Lee et al. (1998). Restriction fragment-length polymorphisms with HaeIII, RsaI and AluI, and sequence analysis of the amplified 16S rDNA, indicated that the phytoplasma in Cuba belonged to the aster yellows group I-A (Arocha et al., 1999). Both ScYLV and ScYP have been detected in samples with symptoms as well as in those without, and coinfection with both organisms is common. It is postulated that the two pathogens were introduced into healthy plants by their respective insect vectors, and henceforth transmitted together by vegetative propagation. No such coexistence is known for other sugarcane diseases. This study, based on the examination of 134 samples obtained from 113 varieties, shows

that both pathogens together are associated with the syndrome in 65 (85´5%) of 76 samples with YLS. It is possible that ScYLV and ScYP can each induce YLS in some varieties under certain conditions, and further investigation is necessary to determine their exact roles. However, failure to detect both pathogens does not preclude symptoms. In this study 11 (48%) of the 23 samples in which neither pathogen could be detected had symptoms (Table 2). This confirms earlier reports that symptoms are not a diagnostically reliable indicator for the presence of either virus or phytoplasma. In another observation, using PCR 46% of 6-month-old symptomless sugarcane were found to be infected with phytoplasma (data not shown). This indicates that the pathogen exists in a large number of varieties, waiting for suitable growth conditions to spread and induce the symptoms. The best option would be to test regardless of symptoms, as they are not completely reliable as to the causal agent involved. The findings that some symptomatic plants failed to show the presence of either pathogen confirm the unreliability of diagnosis based solely on symptomatology, as the methods of detection used here (RT±PCR and nested PCR) are the most sensitive available. However, neither of the two pathogens could be detected in some of the plants that showed symptoms, suggesting either that symptoms similar to YLS were morphologically misdiagnosed in a small proportion of plants, or that the pathogens were present below the limits of detection despite the sensitivity of RT±PCR and nested PCR. Symptoms similar to YLS could also be due to abiotic factors, such as low soil fertility, restricted root growth due to soil compaction, soil-inhabiting fungi and ratooning (Matsuoka & Meneghin, 2000). No pathogen could be expected to be diagnosed in these cases. These abiotic factors should be taken into consideration when assessing YLS in sugarcane. Q 2001 BSPP



The amplification of the 1250 bp phytoplasma 16S rDNA and the 352 bp virus-specific fragments from asymptomatic samples was not unexpected, as substantial latent infection can occur. Symptoms are known to appear in apparently healthy canes within a few days of cold weather or stress. For both pathogens, it seems there is a period of incubation during which they multiply and systemically infect the plant. In some varieties under stress conditions, and despite the presence of both pathogens, no symptoms were observed. This suggests that there is some level of resistance or tolerance in some varieties (Lockhart & CronjeÂ, 2000) which needs to be taken into consideration in managing control of YLS. The results indicate that primers used for the firstround amplification of the phytoplasma, P6 and SN910610, can amplify one group of phytoplasmas. Using these two primers in combination with R16F2n and R16R2, a 1´25 kb specific phytoplasma fragment was amplified. The RFLP analysis of this fragment using RsaI shows that this group is related to the Western-X phytoplasma as described by CronjeÂ & Bailey (1999), based on the classification of Lee et al. (1998). The detection of both pathogens in imported germplasm indicates that the disease may have been spread between countries. The effect of the disease on yield has not been accurately estimated, but figures of 20% loss of recoverable sugar have been reported (Lockhart & CronjeÂ, 2000). Infected plant material can be freed from both virus and phytoplasma by tissue culture (Delage et al., 1999; MSIRI, 1999; MSIRI, 2001). In conclusion, these results indicate that the presence of one or both pathogens is significantly associated with the expression of symptoms. It has also been demonstrated that the detection of YLS based solely on symptoms is totally unreliable because both pathogens are detected in symptomless plants. This could lead to the conclusion that the YLS has probably spread from one country to another because of the mistaken assumption that symptomless plants are free of pathogens, and that heat treatment of cuttings can eliminate them. This is not the case, at least for the phytoplasma.

Acknowledgements We would like to thank Professor Ben Lockhart for providing the anti-ScYLV antiserum and the conjugated antibodies, and Dr Mike Irey for providing the sequences of primers YLS 462 and YLS 111. We are also grateful to Dr Jean Claude Autrey (Director) and Claude Soopramanien (Deputy Director 2 Biology) for helpful criticisms of the manuscript.

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